Genetics: From genes to genomes - Chapter 10: What genes are and what they do

PowerPoint 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*Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display*CHAPTERWhat Genes Are and What They DoCHAPTERPART IIICHAPTER OUTLINECopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10Genomes and Proteomes10.1 Large-Scale Genome Mapping and Analysis10.2 Major Insights from the Human and Model Organism Genome Sequences10.3 Global Analysis of Genes and Their mRNAs10.4 Global Analysis of Proteomes10.5 Repercussions of the Human Genome Project and High-Throughput TechnologyGenomics is the study of whole genomesGenomics involves the development and application of more effective mapping, sequencing, and computational toolsPredict existence and functions of previously undefined genesVerify predictions using molecular biology techniquesCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*The Human Genome Project (HGP)First formally discussed in 1985Discovery science – identification of all elements in a biological systemContrast to hypothesis-driven researchDevelopment of high throughput sequencing technology and computational toolsOfficially began in 1990 – estimated 15 years, $3 billion3-5% of budget committed to studying the ethical, legal, and social implications (ELSI) of human genome mappingSocial and personal repercussions are generating new areas of biological concernCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*DNA sequence coverage of the first rough draft of the human genome2001 - draft sequence (93% of genome) Error rate of 1/10,000 2003 - accurate sequence (97% of genome)2006 – finished sequence (99% of genome) 99.99% accuracyCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Fig. 10.1Number of species with finished whole-genome sequences deposited at the NCBI as of 2/1/2010Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Table 10.1A comparison of the developmental complexity and genome features of model organismsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Table 10.2The information content of an organism's genome is not necessarily proportional to its complexityMaps used in human genome analysisGenomewide linkage mapBased on recombination frequenciesPhysical mapBased on direct analysis of genomic DNAPhysical mapOrdered, overlapping DNA fragments that span each chromosomeHierarchical divide and conquer approachCorrelation between linkage and physical mapHumans, 1 cM ~ 1 Mb; mouse, 1 cM ~ 2 MbCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*The human karyotype: Banding distinguishes the chromosomesCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Fig. 10.2a, bPhotos (upper) and ideograms (lower) of stained human chromosomes at metaphaseAutosomes are numbered in order of descending lengthShort arm is "p"Long arm is "q"Human chromosome 7 at three different levelsFor each chromosome arm, bands and interbands are numbered starting at the centromere and moving out toward the telomereCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Fig. 10.2cFig. 10.4aThe fluorescent in situ hybridization (FISH) protocolCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Preparing chromosome spreads and hybridization of fluorescently-labeled DNA probeThe FISH protocol (cont)Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Fig. 10.4bFig. 10.4a (cont)Visualization of hybridization signals with a fluorescence microscopeNote the four yellow spots where the probe hybridizes to sister chromatids of two homologous chromosomesSequence maps show the order of nucleotides in a cloned piece of DNATwo strategies for sequencing genomes:Hierarchical shotgun approach - used in publicly funded effort to obtain human draft sequenceWhole-genome shotgun approach – used by private company (Celera) to obtain human draft sequenceShotgun approach – randomly generated overlapping insert fragments:Fragments from BACsFragments from shearing whole genomeShearing DNA with sonicationPartial digestion with restriction enzymesCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Idealized representation of the hierarchical shotgun sequencing strategyMake BAC library of genomic DNAGenerate contigs by physical mapping of BAC genomic DNA fragmentsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Fig. 10.5a, bIdealized representation of the hierarchical shotgun sequencing strategy (cont)Chose a set of minimally overlapping BACs to encompass entire genome (minimum tiling path, ~20,000 BACs)Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Fig. 10.5b, cFor each BAC, make plasmid library of ~1000 subclones (~2kb each) and sequence both ends of each insertThen, assemble sequences from each BACHypothetical whole-genome shotgun sequencing strategyCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Fig. 10.6Make three genomic librariesPlasmid library, ~2kb insertsPlasmid library, ~10 kb insertsBAC library, ~200 kb insertsObtain 1000 bp sequence reads from ends of each cloneComputational assembly of sequences into chromosomesNo physical map constructionOnly one BAC libraryRepeats are not a problemMajor insights from human and model organism sequencesTechnological advancesMethods for finding and analyzing genesBiologicalThe human genome contains ~25,000 genesThe genome contains distinct types of gene organizationCombinatorial strategies may lead to gene amplification and diversityGenome sequence studies affirm evolution from a common ancestorCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Advances in gene finding and analysisIdentifying genes in one organism facilitates finding homologous genes in the same or different organismsParalogs – genes that arose by duplication within a single speciesOrthologs – genes in two species that arose from the same gene in a common ancestral speciesExons often encode discrete protein domains that are functional unitsProtein architecture may evolve through creation of different exon combinations (see Fig. 10.7)Blocks of linked loci (syntenic blocks) are found in different species (see Fig. 10.8) Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Expansion of domains and architecture of transcription factors in specific lineagesThe approximate numbers of each domain are shown for each of the three speciesCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Fig. 10.7aUnique and shared domain organization of transcription factors in animalsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Fig. 10.7bSyntenic blocks in the human and mouse genomesDifferent segments of human chromosomes contain at least two genes whose order is conserved in the mouse genomeCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Human chromosomesMouse chromosome keyFig. 10.8The human genome contains ~25,000 genesBefore completion of genome sequence, human genome predicted to have ~100,000 genesIn metazoans, gene number and complexity of organism are not correlatedMechanisms other than gene number must help generate metazoan complexityHumans have a far more complex repertoire of proteins (~1 million different proteins) than other metazoansEvolution of new gene arrangements that alter domain architecture (se Fig. 10.10)Humans have more protein modifications (> 400 different chemical reactions) than other metazoans Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Number of distinct domain architectures in four eukaryotic genomesIn humans, a more complex repertoire of proteins evolved through creation of new gene arrangements that alter domain architectureCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Fig. 10.10The genome contains distinct types of gene organizationGene familiesClosely-related genes (paralogs) that are members of multi-gene familiesCan be clustered together or dispersed on several chromosomesExample in human genome: olfactory receptor (OR) genes arose from multiple duplication events followed by divergence to create 1000 paralogous genes (see Fig 10.11)Other examples – genes that encode histones, hemoglobins (see Chapter 9), immunoglobins, actins, collagens, and heat-shock proteins Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*The genome contains distinct types of gene organization (cont)Gene-rich regionsChromosomal regions that have many more genes than expected from average gene density over entire genomeExample in human genome – class III region of major histocompatibility complex (Fig. 10.12) Gene desertsRegions of >1 Mb that have no identifiable genes3% of human genome is comprised of gene desertsDo they exist simply because the genes are hard to identify (e.g. big genes)?Biological significance of gene-rich regions and gene deserts is not knownCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Class III region of the human major histocompatibility (MHC) complexMHC complex contains 60 genes within a 700 kb regionGC content is much higher than genome averageCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Fig. 10.12Combinatorial strategies at the DNA level may lead to gene amplification and diversityExample – T-cell receptor genes45 variable (V) segments, 2 diversity (D) segments, and 11 joining (J) segmentsIn each T-cell, a different DNA rearrangement creates a complete V-D-J gene (45 x 2 x 11 = 990 combinations)Crucial aspect of immune response to foreign molecules (antigens) Antigen-triggered expansion of T-cell clones that carry particular V-D-J combinationCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Fig. 10.13Combinatorial strategies at the RNA level may lead to gene amplification and diversityExample – three neurexin genesTwo alternative promoters, five sites for alternative splicingCan generate 2000 different mRNAsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Fig. 10.14Genome sequence studies affirm evolution from a common ancestorGenetic components of the basic cellular machinery of all living organisms are remarkably similarAll living organisms are descendants of a single, life-producing biochemistryAnalysis of model organisms can provide biological insights into the corresponding human systemsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Global analysis of genes and their mRNAsDevelopment of technologies to analyze the genome and the complete biochemical machinery of cells were initiated by the Human Genome Project Genomics - global analysis of chromosomal features and gene products Required to gain complete understanding of genome informationHigh throughput instruments - DNA sequencers and DNA arraysPlatforms – all components needed for automated acquisition of dataCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Automated sequencing format using the Sanger sequencing schemeFour sequencing reactions, each has a primer labeled with a different fluorescent dye and a different ddNTPEach synthesized DNA fragment has a different fluorescence based on its terminal ddNTPCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Fig. 10.15aAutomated sequencing format using the Sanger sequencing scheme (cont)Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Fig. 10.15b,dElectrophoresis is done to separate fragments that differ in length by one nucleotide followed by fluorescence scanning of fragmentsAutomated sequencing format using the Sanger sequencing scheme (cont)Data from DNA sequencing experiments are delivered as super-imposed four-color tracesCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Fig. 10.15cAn oligonucleotide arrayDNA arrays have thousands of fragments of known nucleotide sequence spotted at precise locations on a solid supportArrays can be hybridized with fluorescent or radioactive DNA or RNA probesCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Fig. 10.16Two-color DNA microarrays can be used to determine relative expression of genesTwo cDNA samples with different fluorescence labels are mixed together and used as hybridization probes on a DNA arrayCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Fig. 10.17aGreen label for cDNAs from normal yeast cellsRed label for cDNAs from mutant yeast cellsRelative expression levels of ~ 6000 yeast genes on a DNA microarray Red spots represent mRNAs expressed at higher level in mutant cellsGreen spots represent mRNAs expressed at higher levels in normal cellsYellow spots represent mRNAs expressed at equivalent amounts in normal and mutant cellsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Fig. 10.17bAn optical fiber approach to DNA array analysesInstruments have 96 optical fibers of 1 mm diameterThe end of each fiber has 50,000 wellsEach well contains a different oligonucleotide on a beadUsed to interrogate target samples for SNPs or gene expressionCan analyze >106 SNPs a dayCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Fig. 10.18Three types of DNA sequencing used to determine mRNA expression levelsAutomated DNA sequencing of expressed sequence tags (ESTs)Single sequence runs of 600-1000 bp on cDNA insertsSerial analysis of gene expression (SAGE)15 bp from 3' ends of ~ 67 mRNAs linked together into 1000 bp DNA and sequencedQuantitative assessment of thousands of linked tags by DNA sequence analysisMassively parallel signature sequence (MPSS, Fig. 10.19)Most powerful technique for quantifying the transcriptome of individual cells Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Lynx Therapeutics sequencing strategy of multiple parallel signature sequencing (MPSS)106 clones from a cDNA library can be PCR amplified with a unique tagEach amplified product attached to a single beadCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Fig. 10.19MPSS sequencing strategy (cont)Simultaneous sequencing of beads in a flow cells with fluorescent reporter groupsGenerates 106 sequence tags of 17-20 bpGenes identified by comparison of sequence signatures to genome sequenceCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Fig. 10.19 (cont)Global analysis of proteomesProteome – proteins produced by translation of all protein-coding genes from a genomeProteome analysis is highly complexProteins have many variable features that have biological importance and are specific to certain cell types and conditionsEnormous range of expression levels (1-106 copies/cell) for different proteinsNeed to develop analytical methods that can identify and quantify very rare proteins present in complex mixtures of proteinsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Many different features of the proteome to be characterizedGene encoding each proteinChemical modifications to proteinsLevels of expression in different cell types under different conditions (developmental, environmental)Covalent modifications (e.g. phosphorylation, glycosylation)Interactions with other proteins, macromolecules, small moleculesIntracellular compartmentalization (e.g. nucleus, cytoplasm, cell surface)Activation state and half-livesThree-dimensional structureStructure-function relationshipsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*A mass spectrometer allows identification of components in complex mixturesMeasures masses of a wide variety of moleculesMolecules are ionized, transferred to a vacuum, and migration rate in electric field used to estimate massCan sequence amino acids in peptides from fragmentation of proteins in complex mixturesCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Fig. 10.20A strategy using two-dimensional gels and mass spectrometry to identify proteins in complex mixturesPeptide sequences can be identified by comparing mass spectrum against databases of theoretical fragmentsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Fig. 10.21Once peptide sequence is known, gene sequence can be inferred The isotope-coded affinity tag (iCAT) approach to quantifying complex protein mixturesICAT reagent has three componentsBiotin tag – binds tightly to avidin, provides means to purify the peptide of interestLinker with eight hydrogens or eight deuteriums attached to create light and heavy forms of peptidesChemical group that allows attachment of ICAT reagent to all cysteines in a peptideCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Fig. 10.22aCan be used to compare proteins that differ between cells in different states (e.g. normal vs cancer cells)Cells labeled with iCAT reagent (e.g. normal cells, light; cancer cells, heavy) and mixed togetherCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Fig. 10.22bThe iCAT approach to quantifying complex protein mixtures (cont)Proteins purified by avidin affinity chromatography and analyzed by mass spectrometryRelative levels of different proteins present in the two cell types can be determinedCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Fig. 10.22bThe iCAT approach to quantifying complex protein mixtures (cont)Two methods for identification of protein-protein interactionsAffinity capture/mass spectrometry (Fig. 10.23)Capture molecule (e.g. an antibody specific to the protein of interest) is used to "pull down" a protein from a complex mixtureAll proteins associated with the protein of interest are also pulled downProtein arrays (Fig. 10.24)Analogous to DNA arrays, but with protein spotsIdentify substrates for modification (e.g. phosphorylation)Array of antibodies to identify relative levels of proteinsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Using affinity purification and mass spectrometry to identify protein interactionsCells genetically engineered by adding tag sequences to 5' or 3' end of gene encoding the protein of interestAffinity purification based on tag is used to purify complex of proteins that interact with protein of interestProteins in complex identified by mass spectrometryCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Fig. 10.23Protein array of different types of protein kinasesSpectral array of different types of protein kinasesCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Fig. 10.24Radioactivity associated with each kinase after application of radioactive substrateCHiP/chip analyses to identify protein-DNA interactionsCombination of genomic and proteomic approaches to measure protein-DNA interactions required for gene regulatory networksBinding of transcription factors to cis-control DNA elementsBinding of complexes of activators and repressors to DNAChromatin immunoprecipitation (CHiP) Used to identify all genomic sites at which a transcription factor in specific cell types can bind (Fig. 10.25)Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Diagram of the CHiP/chip processCells genetically engineered by adding tag sequences to 5' or 3' end of gene encoding the protein of interestIsolate chromatin from engineered cellsShear chromatin and precipitate protein-DNA complex with anti-tag antibodyPCR-amplify DNA with fluorescent labelUse red for experimental sample and green for control sample prepared from cells that lack tag sequenceHybridize both probes to DNA array (chip) Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Fig. 10.25Repercussions of the Human Genome Project and high-throughput technologyResearch methods for all of biology have been transformedFor each organism, high-throughput platforms can accomplish each of the following tasks:Organism's genome sequenceQuantify the transcriptome/proteome of different cell typesDelineate protein-protein interactions in the proteomeAnalyze other features of the proteome (e.g. subcellular localization, activation, half-life, 3-dimensional structures)Apply genomic approaches to compare cells/organisms with genetic alterations Cancer vs normal cells, knockout vs normal strainsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Great potential exists for predictive/preventive medicineAccess to whole-genome sequencesSequence an individual human genome for < $1000Identify the DNA polymorphisms that are the basis for human variationPredict likelihood of disease in individuals with alleles for polygenic and complex traitsDesign ways to circumvent the limitations of defective genes – e.g. novel drugs, environmental controls, stem cell transplantation, gene therapyCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*Social, ethical, and legal issues have no simple solutionPressing issues raised by individual genome sequencesPrivacy of genetic informationLimitations on the use of genetic testingPatenting of DNA sequencesSocietal views on agingTraining physiciansImpact of increased longevity produced by improved medicineSomatic gene therapy vs germ-line gene therapyFor further descriptions, see the various "Genetics and Society" essays throughout this textbookCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 10*