Genome-wide mutant collectionsSeen in yeast cells and using RNAi in C. ElegansGene knockouts being engineered in every yeast geneHaving this information allows you to SCREEN FOR MUTANTS.RNA interference (RNAi)Method to reduce gene functions in organisms where level of homologous recombination is not high.Protects againstGenome-invading transposable elements and dsRNA virusesIn Plants…known as “co-suppression”DsRNA injection…”RNAi”Nobel Prize winnersCraig Mello and Andy FireApplicationsAnalyze phenotypes by silencing specific genesCreating genome-wide “libraries” of silenced genesControl virus infectionGene therapyLimitations of RNAiNot effective in EVERY cell type ie. In C. Elegans not as effective in nervous system.Sometimes not very stable (subsequent generations not always exhibiting knockout)More of a KNOCKDOWN than KNOCKOUT!!!Might be knocking out more genes than you want to because target gene sequence could be in different spots of the genome.dsRNAmajor proponent of RNAiinitiate ribonuclease protein Dicer which makes siRNAsoaking C. Elegans in solution of dsRNA allows process to occursiRNAsmall interfering RNA20-25 bp long.Added to RISC (RNA-induced silencing complex)Induces cleavage of mRNA template and silences gene expressionInsertional or Chemical MutagenesisRandom mutagenesisMajor ComponentsTransposon insertionsT-DNA insertions in plantsEasy to generate many knockouts in a short amount of timeLarge collection of insertions throughout the genome have been compiled and notedSalk laboratory have mapped nearly every insertion in the Arabidopsis genomeT-DNAComponent of Agrobacterium transformation in plantsTi-plasmid, T-DNA, is transferred into the host plant genome causing tumors in the organism.How it worksInsertion of gene into Ti-plasmid using restriction enzyme and DNA ligase.Introduced into plant cell cultureRegeneration of diseased plant.Transgene that results exhibits a Mendelian Inheritance PatternTo identify a gene with a specific T-DNA insertion…design PCR primers in opposite directions and run a gel to see if the insertion exists.Another way utilizes restriction enzyme digestion to cut the genome at a certain gene site, use DNA ligase to pair it back together into a plasmid…Then use PCR and sequence.Tilling (Targeting Induced Local Lesions in Genomes)Allows direct identification of point mutations in genomesPCR makes fragments of identical length. (Mutant plant will have a mismatch when you do a denaturation/renaturation step)…mutant strands will pair up with wild-type strands…mismatches create kinks in the sequence. Enzyme Cel 1 recognizes a mismatch in DNA and cuts it.Look for fluorescence…if mutation exists there will be 2 bands of different sizes. (size of band determines how far Into the fragment the cut was made…location of mutation.) Scans all genes for mutations.Model OrganismsEasy to grow in labRelatively rapid life cycleGenetic Crosses can be performedLots of offspringHomozygous true-breeding lines can be establishedMutations can be induced easilySmall genomeEasy to transformDNA can be isolated and genes can be clonedYeast (Saccharomyces cerevisiae)Led to discovery of enzymes in glycolytic pathwayLife CycleHaploid and Diploid formsWhy it’s a good modelStable haploid and diploid statesNon-pathogenicEfficient homologous recombinationSmall genome…lecture 16 slide 18Leland Hartwell, Tim Hunt, Paul NurseNobel Prize in Medicine for identifying key genes within the eukaryotic cell cycleIsolating MutantsNon-essential geneMutant phenotype visible in 2 of the 4 haploid spores after meiosisEssential geneOnly two living spores result after meiosis (mutated ones are dead)Temperature-sensitive MutantsPermissive TemperatureNormal Mutant GrowthRestrictive (High) TemperatureMutant protein loses its function.Can see mutant phenotypeUseful in embryonic lethal mutations to allow cells to growYeast cell budsPermissive temperatureDisplays buds of all sizesRestrictive TemperatureShows cells that have completed the cell cycle, but are arrested at a certain point within the cycle.ArabidopsisWhy use?Easy to grow in labSmall size means thousands of plants can be grown in restricted placeRelatively rapid life cycleSelf-fertilizes but can also be out-crossed by hand.Small genome sizeDiploid as opposed to tetraploidy or hexaploidy seen in most plantsMost of the major questions in plant biology can be studied with the Arabidopsis….photosynthesis, plant growth and development, hormones, etc.Large numbers of mutant lines are availableDevelopment of SIMPLE and EFFICIENT transformation methodsRescue mutants by transforming them with wild-type genesAlter genes in vitro then transfer them in vivowww.arabidopsis.orgAgrobacterium dip methodTransformation of T-DNA into a transgenic plant.Mendelian inheritance results as the phenotype is seen in the offspring.Gene GunShooting DNA into cells for transient expression.Non-germline….injection into the somatic cells.Flower DevelopmentFrom Outside to in goes Sepals, Petals, stamens, and carpels in the centersWhorlsDifferent sections within the flower pertaining to the above areas of the flower.Homeotic MutationsForward genetics found these mutations to explain flower development in different plants. (You have the phenotype now you want to clone the sequence to see what gene is mutated)Genes for different mutations were cloned by map-based cloningExpression pattern of cloned genes could be seen by in situ hybridization.Homeotic GenesGenes that say which parts of the body form what body partsMutations to these genes which result in not proper organ development are Homeotic MutationsABC Model of Flower DevelopmentC and A act antagonistically towards one anotherAbsence of one of them indicates that the other will determine the identity of all the floral verticils (sepals and carpels).SepalsCharacterized by A genePetalsCharacterized by co-expression of A and B genesStamensCharacterized by co-expression of B and C genesCarpelsCharacterized by C geneMutation in A gene?Develop Carpels instead of Sepals and Stamens instead of PetalsMutation in B gene? (Pistillata or Apetella 3)Develop Sepals instead of Petals and Carpels instead of StamensEctopic Expression in BOverexpression, so Petals instead of Sepals and Stamens instead of Carpels.Mutation in C gene?Develop Petals instead of Stamens and Sepals instead of Carpels.Mutation of AP1 geneDoes not allow C gene to expand into whorls 1 and 2As a result a bract (leaf)
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