Genetics: From genes to genomes - Chapter 7: What genes are and what they do

*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or displayPowerPoint to accompanyGenetics: From Genes to GenomesFourth EditionLeland H. Hartwell, Leroy Hood, Michael L. Goldberg, Ann E. Reynolds, and Lee M. SilverPrepared by Mary A. BedellUniversity of Georgia*CHAPTERWhat Genes Are and What They DoCHAPTERPART IICHAPTER OUTLINECopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 7Anatomy and Function of a Gene: Dissection Through Mutation7.1 Mutations: Primary Tools of Genetic Analysis7.2 What Mutations Tell Us About Gene Structure7.3 What Mutations Tell Us About Gene Function7.4 A Comprehensive Example: Mutations That Affect VisionMutations: Primary tools of genetic analysisMutations are heritable changes in DNA base sequencesForward mutation – changes wild-type allele to a different allelee.g. A+  a or b+  BReverse mutation (reversion) – changes a mutant allele back to wild typee.g. a A+ or B  b+ Forward mutation rate is usually greater than reversion rate Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Classification of mutations by effect on DNA moleculeSubstitution – replacement of a base by another baseTransition – purine replaced by another purine, or pyrimidine replaced by another pyrimidineTransversion – purine replaced by a pyrimidine, or pyrimidine replaced by a purine Deletion – block of 1 or more bp lost from DNAInsertion – block of 1 or more bp added to DNAInversion – 180° rotation of a segment of DNAReciprocal translocation – parts of two nonhomologous chromosomes change placesCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Mutations classified by their effect on DNACopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Fig. 7.2a - cCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Mutations classified by their effect on DNA (cont)Fig. 7.2dCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Mutations classified by their effect on DNA (cont)Fig. 7.2eRates of spontaneous mutationRates of recessive forward mutations at five coat color genes in mice11 mutations per gene every 106 gametesMutation rates in other organisms2 – 12 mutations per gene every 106 gametes Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Fig. 7.3bDifferent genes, different mutation ratesMutation rates are 10-3 per gene per gameteDifferences in gene sizeSusceptibility of particular genes to various mutagenic mechanismsAverage mutation rate in gamete-producing eukaryotes is higher than that of prokaryotesMany cell divisions take place between zygote formation and meiosis in germ cellsMore chance to accumulate mutationsCan diploid organisms tolerate more mutations than haploid organisms? Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Experimental evidence that mutations in bacteria occur spontaneouslyS. Luria and M. Delbrück (1943) − fluctuation testExamined origin of bacterial resistance to phage infectionInfected wild-type bacteria with phageMajority of cells die, but a few cells can grow and divideHad the cells altered biochemically?Did the cells carry heritable mutations for resistance?Did the mutations arise by chance or did they arise in response to the phage?Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*The Luria-Delbrück fluctuation experimentCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Fig. 7.4Replica plating verifies that bacterial resistanceis the result of preexisting mutationsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Fig. 7.5Interpretation of Luria-Delbrück fluctuation test and replica platingBacterial resistance arises from mutations that occurred before exposure to bactericideBactericide becomes a selective agentKills nonresistant cellsAllows survival of cells with pre-existing resistanceMutations occur as the result of random processesOnce such random changes occur, they usually remain stableCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*How natural processes can change the information stored in DNADepurination 1000/hr in every cellDeamination of C C changed to UNormal C-G  A-T after replicationCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Fig. 7.6a, bHow natural processes can change the information stored in DNA (cont)X-rays break the sugar − phosphate backbone of DNAUltraviolet (UV) light causes adjacent thymines to form abnormal covalent bonds (thymine dimers)Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Fig. 7.6c, dHow natural processes can change the information stored in DNA (cont)Irradiation causes formation of free radicals (e.g. reactive oxygen) that can alter individual bases8-oxodG mispairs with ANormal G-C  mutant T-A after replicationCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Fig. 7.6eMistakes during DNA replication Incorporation of incorrect bases by DNA polymerase is exceedingly rare (< 10-9 in bacteria and humans)Two ways that replication machinery minimizes mistakesProofreading function of DNA polymerase (Fig 7.7)3'-to-5' exonuclease recognizes and excises mismatchesMethyl-directed mismatch repair (later in this chapter)Corrects errors in newly replicated DNACopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*DNA polymerase’s proofreading functionMispaired base is recognized and excised by 3'-to-5' exonuclease of DNA polymeraseImproves fidelity of replication 100-foldCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Fig. 7.7Unequal crossing-over can occur between homologous chromosomesPairing between homologs during meiosis can be out of registerUnequal crossing-over results in a deletion on one homolog and a duplication on the other homologCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Fig. 7.8aTransposable elements (TEs) move around the genomeTEs can "jump" into a gene and disrupt its functionTwo mechanisms of TE movement (transposition)Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Fig. 7.8bExperimental evidence that mutagens induce mutationsH. J. MullerX-ray dose above the naturally-occurring level causes increased mutation rate in DrosophilaExposed male Drosophila to X-raysMating scheme (see Fig 7.9) used genetically marked "balancer" X chromosomeAble to detect X-linked genes that are essential for viabilityCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Exposure to X-rays increases the mutation rate in DrosophilaCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Fig. 7.9How mutagens alter DNA:Chemical action of mutagenReplace a base: Base analogs - chemical structure almost identical to normal baseCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Fig. 7.10aHow mutagens alter DNA:Chemical action of mutagenCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Alter base structure and properties: Hydroxylating agents add an –OH groupFig. 7.10bHow mutagens alter DNA:Chemical action of mutagen (cont)Alter base structure and properties (cont): Alkylating agents add ethyl or methyl groupsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Fig. 7.10bHow mutagens alter DNA:Chemical action of mutagen (cont)Alter base structure and properties (cont): Deaminating agents remove amine (-NH2) groupsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Fig. 7.10bHow mutagens alter DNA:Chemical action of mutagen (cont.)Insert between bases: Intercalating agentsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Fig. 7.10cDNA repair mechanisms that are very accurateReversal of DNA base alterationsAlkyltransferase – removes alkyl groupsPhotolyase – splits covalent bond of thymine dimersHomology-dependent repair of damaged bases or nucleotidesBase excision repair (Fig 7.11)Nucleotide excision repair (Fig 7.12)Correction of DNA replication errorsMethyl-directed mismatch repair (Fig 7.13)Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Base excision repair removes damaged basesDifferent glycosylases cleave specific damaged basesParticularly important for removing uracil (created by cytosine deamination) from DNACopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Fig. 7.11Nucleotide excision repair corrects damaged nucleotidesUvrA – UvrB complex scans for distortions to double helix (e.g. thymine dimers)UvrB – UvrC complex nicks the damaged DNA 4 nt to one side of damage7 nt to the other side of damageCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Fig. 7.12In bacteria, methyl-directed mismatch repaircorrects mistakes in replicationParental DNA strand marked by adenine methylase Methyl group added to A in GATC sequenceNewly-replicated DNA isn't yet methylatedMutS and MutL bind to mismatched nucleotidesMutH nicks the unmethylated strand opposite the methylated GATCCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Fig. 7.13In bacteria, methyl-directed mismatch repaircorrects mistakes in replication (cont)Gap made in unmethylated (new) strand by DNA exonucleasesGap filled in by DNA polymerase using the methylated (old) strand as templateCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Fig. 7.13 (cont)DNA repair mechanisms that are error-proneSOS system – bacteriaUsed at replication forks that stalled because of unrepaired DNA damage"Sloppy" DNA polymerase used instead of normal polymeraseAdds random nucleotides opposite damaged basesNonhomologous end-joining (Fig 7.14)Deals with double-strand DNA breaks caused by X-rays or reactive oxygenCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Repair of double-strand breaks by nonhomologous end-joiningCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Fig. 7.14Unrepaired double-strand breaks can lead to lethal chromosome rearrangements (e.g. deletions, inversions, translocations)Resection step can lead to loss of DNAHealth consequences of mutations in genes encoding DNA repair enzymesXeroderma pigmentosum:Mutations in one of seven genes encoding enzymes involved in nucleotide excision repairCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Fig. 7.15Hereditary forms of colorectal cancer (not shown):Mutations in human homologs of bacterial genes (MutS and MutL) involved in mismatch repairImpact of unrepaired mutationsGerm line mutations – occur in gametes or in gamete precursor cellsTransmitted to next generationProvide raw material for natural selectionSomatic mutations – occur in non-germ cellsNot transmitted to next generation of individualsCan affect survival of an individualCan lead to cancerCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Fig. 7.16The Ames test identifies potential carcinogensAssay uses his- mutants in S. typhimuriumDetects mutations that cause his- to his+ reversionWhat mutations tell us about gene structureComplementation testingReveals whether two mutations are in a single gene or in different genes"Complementation group" is synonymous with a geneFine structure mapping Seymour Benzer used phage T4 mutantsExperimental evidence that a gene is a linear sequence of nucleotide pairsSome regions of chromosomes have "hot spots" for mutationsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Drosophila eye color mutations produce a variety of phenotypesDo these phenotypes result from allelic mutations or from mutations in different genes? Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Fig. 7.17Complementation testing of Drosophila eye color mutationsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Fig. 7.18aA complementation table for X-linked eye color mutations in DrosophilaThese results reveal five complementation groups (genes):Mutations in white, cherry, coral, apricot, and buff are allelic [(all affect the white (w) gene]Mutations in garnet, ruby, vermillion, and carnation are not allelic with each other or with white mutationsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Fig 7.18bFine structure mapping of phage T4 mutantsSeymour Benzer (mid 1950-1960s)Can recombination take place between different mutations in the same gene (intragenic recombination)?Phage T4 is a virus that infects E. coliAdvantages of phage T4 for fine structure mapping: Each phage can produce 100-1000 progeny in <1 hrEasy to produce large numbers of progeny to detect rare eventsConditions allowed proliferation of only recombinant phages and death of parental phagesCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*How recombination within a gene couldgenerate a wild-type alleleCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Fig 7.19Working with bacteriophage T4Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Fig 7.20a(a.1)(a.3)(a.2)Counting bacteriophages by serial dilutionCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Fig 7.20a(a.4)Phenotypic properties of rII – mutants of bacteriophage T4 (cont)rII – mutants have an altered plaque morphology and altered host rangeCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Fig 7.20bBenzer's experimental approach to fine structure mapping of the rII locusGenerated 1612 spontaneous point mutations and several deletions in rII locusIdentified two complementation groups, rIIA and rIIB (Fig 7.20c)Mapped locations of deletions relative to each other using recombination (Fig 7.21a)Mapped locations of point mutations relative to the deletions (Fig 7.21a)Tested for recombination between all point mutations within the same complementation group (Fig 7.20d)Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*A customized complementation test between rII – mutants of bacteriophage T4Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*(c.1) Complementation test (trans configuration)(c.2) Control(cis configuration)Fig 7.20cDetecting recombination between two allelic mutationsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Fig 7.20d(d.1) Recombination test (d.2) ControlUsing deletions for rapid mappingCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Fig 7.21aFine structure of the phage T4 rII regionCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Fig 7.21cMutation hotspots suggest that some nucleotides are more susceptible to mutations than othersWhat mutations tell us about gene functionGarrod (1902) – some human diseases result from "inborn errors of metabolism" (Fig 7.22)Beadle and Tatum (1940s) – "the one gene, one enzyme" hypothesis (Fig 7.23)Neurospora crassa, mutants in arginine (arg) synthesisGenetic dissection of a biochemical pathwayIngram (mid-1950s) – mutations in a gene can result in amino acid substitutions that disrupt the function of the encoded proteinMissense substitution in hemoglobin β causes sickle cell anemia Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Alkaptonuria: An inborn error of metabolismCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Fig 7.22Beadle and Tatum: The "one gene, one enzyme" hypothesisScreened for X-ray induced mutations in Neurospora that disrupted synthesis of arginine (arg)Prototroph – wild-type strain that grows in minimal media without nutritional supplementsAuxotroph – mutant strain that cannot grow in minimal mediaRecombination analysis used to map mutations to four different regions of genomeEach region contained a different complementation groupFour genes for arg biosynthesis – ARG-E, ARG-F, ARG-G, and ARG-HCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Experimental support for the “one gene, one enzyme” hypothesisScheme used by Beadle and Tatum for isolation of arg – auxotrophs in NeurosporaCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Fig 7.23aExperimental support for the “one gene, one enzyme” hypothesisGrowth response if nutrient is added to minimal mediumInferred biochemical pathwayEach ARG gene encodes an enzyme needed to convert one intermediate to the next in the pathwayCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Fig 7.23b, cProteins are chains of amino acidslinked by peptide bonds20 different amino acidsR group is the side chain that is unique to each amino acidFour groups of amino acids based on R group properties (Fig 7.24b)–COOH group and –NH2 group of adjacent amino acids are joined in covalent peptide bondPolypeptides have "N terminus" and "C terminus"Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Fig 7.24a, cCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Fig 7.24bAmino acids with nonpolar R groupsR groupsBackboneR groupsBackboneAmino acids with uncharged R groupsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Fig 7.24b (cont)R groupsBackboneR groupsBackboneAmino acids with charged R groupsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Basic R groupAcidic R groupFig 7.24b (cont)R groupsBackboneR groupsBackboneR groupsBackboneR groupsBackboneThe molecular basis of sickle-cell anemiaGluVal substitution at sixth amino acid affects the three-dimensional structure of the hemoglobin b chainAbnormal protein aggregates cause sickle shape of red blood cellsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Fig 7.25aSickle-cell anemia is pleiotropicCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Fig 7.25bLevels of polypeptide structureInteractions that determine the three-dimensional conformation of a polypeptideCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Fig 7.26aLevels of polypeptide structure (cont)Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*1o structure is the amino acid sequence2o structure is the characteristic geometry of localized regionsFig 7.26b, cLevels of polypeptide structure (cont)Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*3o structure is the complete three-dimensional arrangement of a polypeptideFig 7.26dMultimeric proteins are complexes of polypeptide subunitsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Identical subunitsNon-identical subunitsFig 7.27a, bMultimeric proteins are complexes of polypeptide subunits (cont)Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*One polypeptide in different proteinsFig 7.27cMultimeric proteins are complexes of polypeptide subunits (cont)Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Microtubules: large assemblies of subunitsFig 7.27dOne gene, one polypeptide"One gene, one enzyme" concept is not broad enoughNot all proteins are enzymesSome proteins are multimeric and subunits are encoded by different genesComplex pathways can be dissected through genetic analysisDifferent mutations in a single gene can produce different phenotypesDifferent amino acid substitutions can have different effects on protein functionMutations can affect protein function by altering the amount of normal protein madeCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*A comprehensive example: Mutations that affect visionCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Fig 7.28The cellular basis of visionThe molecular basis of visionHow mutations modulate light and color perceptionCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Fig 7.29a - cUnequal crossing-over between red and green photoreceptor genes can change gene number and create hybrid photoreceptor proteinsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th ed., Chapter 7*Fig 7.29d