PowerPoint PresentationSlide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Slide 25Slide 26Slide 27Slide 28Slide 29Slide 30Slide 31Slide 32Slide 33Slide 34Slide 35Slide 36Slide 37Slide 38Slide 39Slide 40Slide 41Slide 42Slide 43Figure 15-20Figure 15-21Slide 46Figure 15-5Lecture 23October 22, 2014Part I: Translation (continued) Part II:Introduction to Genetic Analysis (Ch. 14)•Forward and reverse genetics•Identifying biochemical pathways using mutations•(Biochemical genetics)Part II: Mutations I (Ch. 15)Ingredients for translation - II : The prokaryotic (70S) ribosomePeptide exit channelA = Aminoacyl siteP = Peptidyl siteE = Exit siteCryo-electron microscopyTranslation elongation (Prokaryotes)GTP-hydrolysisGTP- hydrolysisEF-G leavestRNA in theE(xit) site leaves with EF-Tu4 StepsTranslation: Multiple ribosomes on one mRNA = polysome = polyribosomeTranslation termination (Prokaryotes)Ribosome disassembles and releases mRNA.Release factor (protein) resembles a tRNA.RF cleaves the peptide from the tRNA.Translation termination – premature 1. Nonsense mutation:A regular codon is mutated to a stop-codon.= premature stop codon -> loss of function of protein2. Suppressor mutation:A secondary mutation in a tRNA gene allows the tRNA to recognize a stop codon.= stop codon readthrough -> regain of functionAUG StopExample: Cystic fibrosis transmembrane conductance regulator (CFTR)'Nonsense' - mediated mRNA decayOpen reading frameWild type mRNAAUG StopOpen reading frameExon-exon splice junctionExon-junctionprotein complexWild type proteinUGGTrpUGAStopTruncated open reading frameDisease allele with ‘Nonsense’mutationUGGTrpTruncated proteinTruncated protein AND unstable mRNA -> DiseasemRNA decayby ribonuclease********Example: Cystic fibrosis transmembrane conductance regulator (CFTR)Nonsense-mediated mRNA decayUGAtRNADrug XAmino acidSubstantially functional proteinXmRNAstabilizedExon-exon splice junctionExon-junctionprotein complexDrug screening concept: Look for drugs that suppress termination /allow Stop-codon ‘readthrough’.UGAStopmRNA decayTruncated open reading frameDisease allele with ‘Nonsense’mutationby ribonucleaseTruncated protein-> Disease********Introduction to Genetic AnalysisInteraction of genes in pathways: Biochemical geneticsMetabolic disordersBeadle and TatumOne gene one enzyme hypothesisMutant screen / Forward genetic analysisHeritable defects in phenylalanine metabolismArchibald Garrod, M.D.Proposed in 1902 that Alkaptonuria is due to a recessive allele. A brief history of PKU research1934: Folling (Norway) describes PKU syndrome1947: PKU ascribed to defect in Phe hydroxylase -> Dietary restrictions (casein protein) ameliorate PKU symptoms1961: Newborn testing begins (Guthrie)1967: 37 states’ laws require newborn testing1983: Woo identifies human ‘PKU gene’. -> Genetic testing is possible. Biochemical Pathway**PhenylketonuriaOverview: Introduction to Forward Genetic Analysis• Identify a question to be addressed• Generate a collection of mutant strains - Mutagenesis• Screen the collection for mutant phenotypes• Describe the mutant phenotypes-> Learn what the wild-type allele does • Study genetic interactions (e.g. epistasis)-> Order the genes into a pathway• Isolate the gene = Molecular cloning -> Mechanism --> Ornithine --> Citrulline --> Argininearg-3arg-1 arg-2The Arginine biosynthesis pathway of NeurosporaGeorge Beadle and Ed Tatum 1940’sAmerican Museum of Natural History NYWT Yes Yes (+) Yes (+)arg-1** No No (-) Yes (+) *ArgininesynthesisGrowthwithout arginine with arginine* Supplementation with arginine rescues (complements) the mutant growth defect.** arg-1 is an ‘auxotroph’ (“ arg minus”). Working with mutant strainsGenotype PhenotypeIntroduction to Genetic Analysis I - Mutagenesis Example: Beadle and Tatum (1940s) Biochemical genetics of NeurosporaIntroduction to Genetic Analysis II - Mutant screenPrimary screenSecondary screenIntroduction to Genetic Analysis III - Phenotypic characterizationThe mutant is arg- (“arg minus”)Naming the mutant: arg-1. Wild type: arg-1+Phenotypic characterization of three arg- mutantsOverview: Introduction to Forward Genetic Analysis• Identify a question to be addressed• Generate a collection of mutant strains - Mutagenesis• Screen the collection for mutant phenotypes• Describe the mutant phenotypes-> Learn what the wild-type allele does • Study genetic interactions (e.g. epistasis)-> Order the genes into a pathway --> Ornithine --> Citrulline --> Argininearg-3arg-1 arg-2“1 Gene -> 1 Enzyme - Hypothesis”Beadle and TatumQ: What is the phenotype of an arg-2, arg-3 double mutant? Like arg-2? Like arg-3?Q: Are arg-1 and arg-2 'allelic' mutations (in the same gene)?Summary: Introduction to Genetic AnalysisForward Genetics Reverse Genetics• Identify the question (biochemistry)• Generate a mutant collection• Screen for mutations• Mutant characterization• Genetic pathway• Identify the genes (DNA level)•Infer physiological role•What is wrong in mutants?•Infer biochemical function•Pick a gene (DNA)MutationA change in the base pair sequence of DNA•Types of Mutations•Cells affected by mutations•Effects of mutations•Causes of mutationsDNA repairMutationsTypes of mutations (at the DNA level)Single-gene mutation Single-nucleotide substitutions (Point mutations)Insertions and deletions (Indels)Chromosome mutations Mutations in chromosome numberCells affected by mutationGermline cellsMutation passed on to offspringSomatic cellsonly inherited to daughter cells (clone)e.g. may lead to cancerCauses of mutationsAgents:• Physical• Chemical• Biological‘Induced’ (mutagens) versus ‘Spontaneous' Effects of mutationsAt the protein levelAt the level of gene expressionDNA RepairRepair of bases e.g. Thymidine dimer repairRepair of nucleotides, e.g. Excision repairExample: C->T(frequent)Example: C->A(rare)Types of mutations1. Single-base substitutions2. Small Insertions/deletionsWhat is the effect of mutations in introns and 5’ upstream regions?Silent?Effects of mutations at the molecular (protein) level Function?Neutral?Reduced?Increased?Loss?Loss?Loss?Effects of mutations in non-coding regionsWhat is the most likely effect on gene function in each case?1.