10 12 2012 13 42 00 Genome wide mutant collections Seen in yeast cells and using RNAi in C Elegans Gene knockouts being engineered in every yeast gene Having 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 against o Genome invading transposable elements and dsRNA viruses In Plants known as co suppression DsRNA injection RNAi Nobel Prize winners o Craig Mello and Andy Fire Applications o Analyze phenotypes by silencing specific genes o Creating genome wide libraries of silenced genes o Control virus infection o Gene therapy Limitations of RNAi o Not effective in EVERY cell type ie In C Elegans not as effective in nervous system o Sometimes not very stable subsequent generations not always exhibiting knockout o More of a KNOCKDOWN than KNOCKOUT o Might be knocking out more genes than you want to because target gene sequence could be in different spots of the genome dsRNA major proponent of RNAi initiate ribonuclease protein Dicer which makes siRNA soaking C Elegans in solution of dsRNA allows process to occur small interfering RNA siRNA 20 25 bp long Added to RISC RNA induced silencing complex Induces cleavage of mRNA template and silences gene expression Insertional or Chemical Mutagenesis Random mutagenesis Major Components o Transposon insertions o T DNA insertions in plants Easy to generate many knockouts in a short amount of time Large collection of insertions throughout the genome have been compiled and noted Arabidopsis genome o Salk laboratory have mapped nearly every insertion in the T DNA Component of Agrobacterium transformation in plants o Ti plasmid T DNA is transferred into the host plant genome causing tumors in the organism How it works o Insertion of gene into Ti plasmid using restriction enzyme and DNA ligase o Introduced into plant cell culture o Regeneration of diseased plant Transgene that results exhibits a Mendelian Inheritance Pattern To 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 genomes PCR 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 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 cuts it mutations Model Organisms Easy to grow in lab Relatively rapid life cycle Genetic Crosses can be performed Lots of offspring Homozygous true breeding lines can be established Mutations can be induced easily Small genome Easy to transform DNA can be isolated and genes can be cloned Yeast Saccharomyces cerevisiae Led to discovery of enzymes in glycolytic pathway Life Cycle o Haploid and Diploid forms Why it s a good model o Stable haploid and diploid states o Non pathogenic o Efficient homologous recombination o Small genome lecture 16 slide 18 Leland Hartwell Tim Hunt Paul Nurse Nobel Prize in Medicine for identifying key genes within the eukaryotic cell cycle Isolating Mutants Non essential gene meiosis Essential gene o Mutant phenotype visible in 2 of the 4 haploid spores after o Only two living spores result after meiosis mutated ones are dead Temperature sensitive Mutants o Permissive Temperature Normal Mutant Growth o Restrictive High Temperature Mutant protein loses its function Can see mutant phenotype o Useful in embryonic lethal mutations to allow cells to grow Yeast cell buds Permissive temperature o Displays buds of all sizes Restrictive Temperature o Shows cells that have completed the cell cycle but are arrested at a certain point within the cycle Arabidopsis Why use o Easy to grow in lab o Small size means thousands of plants can be grown in restricted place o Relatively rapid life cycle o Self fertilizes but can also be out crossed by hand o Small genome size o Diploid as opposed to tetraploidy or hexaploidy seen in most Most of the major questions in plant biology can be studied with the Arabidopsis photosynthesis plant growth and development plants hormones etc Large numbers of mutant lines are available Development of SIMPLE and EFFICIENT transformation methods o Rescue mutants by transforming them with wild type genes o Alter genes in vitro then transfer them in vivo o www arabidopsis org Agrobacterium dip method Transformation of T DNA into a transgenic plant Mendelian inheritance results as the phenotype is seen in the offspring Gene Gun Shooting DNA into cells for transient expression Non germline injection into the somatic cells Flower Development From Outside to in goes Sepals Petals stamens and carpels in the centers Whorls o Different sections within the flower pertaining to the above areas of the flower o Homeotic Mutations Forward 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 Expression pattern of cloned genes could be seen by in based cloning situ hybridization Homeotic Genes Genes that say which parts of the body form what body parts Mutations to these genes which result in not proper organ development are Homeotic Mutations ABC Model of Flower Development C and A act antagonistically towards one another o Absence of one of them indicates that the other will determine the identity of all the floral verticils sepals and carpels Sepals o Characterized by A gene Petals Stamens Carpels o Characterized by co expression of A and B genes o Characterized by co expression of B and C genes o Characterized by C gene Mutation in A gene Develop Carpels instead of Sepals and Stamens instead of Petals Mutation in B gene Pistillata or Apetella 3 Develop Sepals instead of Petals and Carpels instead of Stamens Ectopic Expression in B Overexpression so Petals instead of Sepals and Stamens instead of Carpels Mutation in C gene Develop Petals instead of Stamens and
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