Genetics: From genes to genomes - Chapter 16: How genes are regulated

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 Hartwell et al., 4th editionHow Genes Are Regulated*PART VCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16Gene Regulation in Eukaryotes16.1 Overview of Eukaryotic Gene Regulation16.2 Control of Transcription Initiation16.3 Chromatin Structure and Epigenetic Effects16.4 Regulation After Transcription16.5 A Comprehensive Example: Sex Determination in DrosophilaCHAPTER OUTLINECHAPTEROverview of eukaryotic gene regulationEukaryotes use complex sets of interactionsRegulated interactions of large networks of genesEach gene has multiple points of regulationThemes of gene regulation in eukaryotes:Environmental adaptation in unicellular eukaryotesMaintenance of homeostasis in multicellular eukaryotesGenes are turned on or off in the right place and timeDifferentiation and precise positioning of tissues and organs during embryonic developmentCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Compared to prokaryotes, eukaryotes have additional levels of complexity for controlling gene expressionEukaryotic genomes are larger than prokaryotic genomesChromatin structure in eukaryotes makes DNA unavailable to transcription machineryAdditional RNA processing events occur in eukaryotesIn eukaryotes, transcription takes place in the nucleus and translation takes place in the cytoplasmCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Key regulatory differences between eukaryotes and prokaryotesCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Table 16.1Multiple steps where production of the final gene product can be regulated in eukaryotes Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Fig. 16.1Control of transcription initiationThree types of RNA polymerases in eukaryotesRNA pol I – transcribes rRNA genesRNA pol II – transcribes all protein-coding genes (mRNAs) and micro-RNAsRNA pol III – transcribes tRNA genes and some small regulatory RNAsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*RNA polymerase II transcriptionRNA pol II catalyzes synthesis of the primary transcript, which is complementary to the template strand of the geneMost RNA pol II transcripts undergo further processing to generate mature mRNARNA splicing – removes intronsAddition of 5' GTP cap – protects RNA from degradationCleavage of 3' end and addition of 3' polyA tail Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*cis-acting elements: promoters and enhancersPromoters – usually directly adjacent to the geneInclude transcription initiation siteOften have TATA box:Allow basal level of transcription Enhancers – can be far away from geneAugment or repress the basal level of transcriptionCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Fig. 16.2Use of reporter genes to identify promoters and enhancers in eukaryotesExamples of reporter genes used in eukaryoteslacZ gene (Ch 15), blue color when X-gal usedGene for "green fluorescent protein" (GFP)Mutations in regulatory regions can be engineered and tested for effects on expression Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Fig. 16.3trans-acting factors interact with cis-acting elements to control transcription initiationCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Fig. 16.4Direct effects of transcription factors: Through binding to DNAIndirect effect of transcription factors:Through protein-protein interactions Use of reporter genes to identify trans-acting factors in transcriptional regulationCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Fig. 16.5Basal transcription factorsBasal transcription factors assist the binding of RNA pol II to promoters (see Fig 16.6)Key components of basal factor complex:TATA box-binding protein (TBP)Bind to TATA boxFirst of several proteins to assemble at promoter TBP-associated factors (TAFs)Bind to TBP assembled at TATA boxRNA pol II associates with basal complex and initiates basal level of transcriptionCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Basal factors bind to promoters of allprotein-encoding genesCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Fig. 16.6Ordered pathway of assembly at promoter:1. TBP binds to TATA box2. TAFs bind to TBP3. RNA pol II binds to TAFsActivators are transcription factors that bind to enhancersActivators are responsible for much of the variation in levels of transcription of different genesIncrease levels of transcription by interacting directly or indirectly with basal factors at the promoter3-dimensional complex of proteins and DNA (Fig. 16.7)Mechanisms of activator effects on transcriptionStimulate recruitment of basal factors and RNA pol II to promotersStimulate activity of basal factors already assembled on promotersFacilitate changes in chromatin structureCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Binding of activators to enhancers increases transcriptional levelsLow level transcription occurs when only basal factors are bound to promoterCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*When basal factors and activators are bound to DNA, rate of transcription increasesFig. 16.7Domains within activatorsActivator proteins have two functional domainsSequence-specific DNA binding domain (Fig 16.8)Binds to enhancerTranscription-activator domainInteracts with other transcriptional regulatory proteinsSome activators have a third domain (Fig 16.9)Responds to environmental signalsExample - steroid hormone receptorsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*DNA-binding domains of activator proteinsInteracts with major groove of DNASpecific amino acids have high-affinity binding to specific nucleotide sequenceThe three best-characterized motifs:Helix-loop-helix (HLH)Helix-turn-helix (HTH)Zinc fingerCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Fig. 16.8Steroid hormone receptors are activators only in the presence of specific hormonesSteroid hormones don't bind to DNA but are coactivators of steroid hormone receptors In the absence of hormone, these receptors cannot bind to DNA and so cannot activate transcriptionIn the presence of hormone, these receptors bind to enhancers for specific genes and activate their expressionCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Fig. 16.9Many activators must form dimers to functionHomodimers: multimeric proteins made of identical subunitsHeterodimers: multimeric proteins made of nonidentical subunitsExamples - Fos and Jun, both have leucine zippersFos forms heterodimers with Jun, but cannot form homodimersJun can form homodimersJun-Jun and Jun-Fos dimers bind to the same enhancer sequence, but have different affinitiesCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Fig. 16.10Dimerization domains make up another class of transcription factor domainsDimerization domains are specialized for polypeptide-polypeptide interactions Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Fig. 16.11Leucine zippers are a common dimerization motif in eukaryotesAmino acid sequence twirls into an a helix with leucines protruding at regular intervals Repressor proteins suppress transcription initiation through different mechanismsSome repressors have no effect on basal transcription but suppress the action of activatorsCompete with activator for the same enhancer (Fig 16.12a) ORBlock access of activator to an enhancer (Fig 16.12b)Some repressors eliminate virtually all basal transcription from a promoterBlock RNA pol II access to promoter ORBind to sequences close to promoter or distant from promoter Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Repressor proteins that act through competition with an activator proteinRepressor binds to the same enhancer sequence as the activatorHas no effect on the basal transcription levelCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Fig. 16.12aRepressor proteins that act through quenching an activator proteinQuenchers bind to the activator but do not bind to DNAType I: Repressor blocks the DNA-binding domainType II: Repressor blocks the activation domain Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Fig. 16.12bThe same transcription factor can playdifferent roles in different cellsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Fig. 16.13Example: a2 repressor in yeast Determines mating type by binding to enhancers of certain genesIn a haploids and a/a diploids, a2 binds to enhancers of a-specific genes but not to enhancers of haploid-specific genesIn a/a diploids, binding specificity of a2 is altered so that it can bind to enhancers of haploid-specific genes The Myc-Max mechanism can activate or repress transcriptionMyc − identified as an oncogene Regulates transcription of genes involved in cell proliferationDoesn't have DNA binding activity on its ownMax − identified through its binding to MycMax/Max homodimers and Myc/Max heterodimers bind to the same enhancersMax/Max represses transcriptionMyc/Max activates transcriptionCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Comparative structures of Myc and MaxMyc/Myc homodimers cannot formMax/Max homodimers and Myc/Max heterodimers can formMax/Max homodimers bind DNA but cannot act as activatorsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Fig. 16.14Dimer structure and subunit concentrations of Myc and Max affect activation or repressionMax is constitutively expressed but Myc expression is tightly regulatedMax/Max acts as a repressor when Myc is not expressedCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Fig. 16.15aDimer structure and subunit concentrations of Myc and Max affect activation or repressionWhen Myc is present, Myc/Max heterodimers form and activate transcriptionMyc - Max affinity is higher than Max - Max affinityCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Fig. 16.15bComplex regulatory regions enable fine-tuning of gene expressionEach gene can have many regulatory proteinsIn humans, ~2000 genes encode transcriptional regulatory proteinsEach regulatory protein can act on many genesEach regulatory region can have dozens of enhancersEnhanceosome – multimeric complex of proteins and other small molecules that associate with an enhancerEnhancers can be bound by activators and repressors with varying affinitiesDifferent sets of cofactors and corepressors compete for binding to activators and repressorsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Different enhancers for the string gene in Drosophila are used in different cell types Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Fig. 16.16Cells in different parts of Drosophila embryos go through the 14th mitosis at different timesThe string gene product activates the 14th mitosisDifferent activators for string are expressed at different times in different tissuesChromatin structure and epigenetic effectsChromatin structure can affect transcriptionNucleosomes can sequester promoters and make them inaccessible to RNA polymerase and transcription factorsHistone modification and DNA methylationChromatin remodeling and hypercondensationEpigenetic changes – changes in chromatin structure that are inherited from one generation to the nextDNA sequence is not alteredCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Chromatin reduces transcriptionCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Fig. 16.17Effects of chromatin structure on transcription: Histone modification and DNA methylationN-terminal tails of histones H3 and H4 can be modifiedMethylation, acetylation, phosphorylation, and ubiquitinationCan affect nucleosome interaction with other nucleosomes and with regulatory proteinsCan affect higher-order chromatin structureDNA methylation occurs at C5 of cytosine in a CpG dinucleotideAssociated with gene silencing Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Determining methylation state of DNA using two restriction enzymes and Southern blottingHpaII and MspI have the same recognition sequence (CCGG), but different sensitivity to DNA methylationCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Fig. 16.18HpaII doesn't cut if 2nd C is methylatedMethylation doesn't affect MspI cuttingChromatin remodeling can expose the promoterNucleosomes can be repositioned of removed by chromatin remodeling complexesAfter remodeling, DNA at promoters and enhancers becomes more accessible to transcription factorsCan be assayed using DNase digestion (DNase hypersensitive sites)Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Fig. 16.19The SWl-SNF remodeling complexOne of best-studied remodeling complexesUses energy from ATP hydrolysis to alter nucleosome positioningCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Fig. 16.20Hypercondensation of chromatin results in silencing of transcriptionExamples of heterochromatin – inactive X chromosome, centromeres, telomeresHeterochromatin has methylation of CpG dinucleotides and methylation of lysine in histone H3 tailsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Fig. 16.21Gene silencing by the SIR complex in yeastGene for a2 repressor is at the MAT (mating type) locusHML and HMR are copies of MAT locus but are silenced by SIR complexCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*SIR mutations cause sterility because HML and HMR genes not silenced and both a- and a-specific genes are expressedFig. 16.22Genomic imprinting results from transcriptional silencingGenomic imprinting – expression of a gene depends on whether it was inherited from the mother or fatherOccurs with some genes of mammalsEpigenetic effect (no change in DNA sequence)Paternally imprinted gene is transcriptionally silenced if it was transmitted from the fatherMaternal allele is expressedMaternally imprinted gene is transcriptionally silenced if it was transmitted from the motherPaternal allele is expressedCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*In mice, deletion of Igf2 causes a mutant phenotype only when transmitted by fatherMaternal imprinting of Igf2 geneMaternally-inherited Igf2 allele is silencedIgf2 deletion heterozygotes have normal phenotype if the mutant allele was inherited from the motherCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Fig. 16.23aMethylation of complementary strands of DNA in genomic imprintingEpigenetic state can be maintained across cell generations through the action of DNA methylasesCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Fig. 16.23bInsulators limit the chromatin region over which an enhancer can operateAn insulator that binds CTCF is involved in reciprocal imprinting of the Igf2 and H19 genes Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Fig.16.23cMethylation of the H19 promoter and the insulator occurs in spermatogenesis A methylase is essential for imprinting of the H19 gene The resetting of genomic imprints during meiosisEpigenetic imprints remain throughout the lifespan of the mammalIn germ cells, epigenetic imprints are reset at each generationDuring meiosis, imprints are erased and new ones are setCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Fig. 16.23dGenomic imprinting and human diseaseExamples: two syndromes associated with small deletions in chromosome 15At least two genes within this region are differently imprintedPraeder-Willi syndrome occurs when the deletion is inherited from the fatherAngelman syndrome occurs when the deletion is inherited from the motherAffected individuals have mental retardation and development disordersCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Inheritance patterns of disorders resulting from mutations in imprinted genesThese pedigrees may appear to be instances of incomplete penetrance, but are distinctly differentCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Fig. 16.23eRegulation after transcriptionPosttranscriptional regulation can occur at any stepAt the level of RNA Splicing, stability, and localizationExample – alternative splicing of mRNAGenerates more diversity of proteinsCommon feature in eukaryotesAt the level of proteinSynthesis, stability, and localizationCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Expression of the Sxl mRNA in early Drosophila developmentSex lethal (Sxl) gene encodes a protein required for female-specific development (see Comprehensive example)In early embryos, Sxl is transcribed only in femalesCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Fig. 16.24aDifferential RNA splicing of Sxl mRNA during Drosophila developmentLater in development, Sxl gene is transcribed in both sexesSxl protein regulates alternative splicing of its own mRNACopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Fig. 16.24bSome small RNAs are responsible for RNA interference (RNAi)Specialized RNAs that prevent expression of specific genes through complementary base pairingSmall (21 – 30 nt) RNAs Micro-RNAs (miRNAs) and small interfering RNAs (siRNAs)First miRNAs (lin-4 and let-7) identified in C. elegansNobel prize to A. Fire and C. Mello in 2006Posttranscriptional mechanisms for gene regulationmRNA stability and translationMay also affect chromatin structureCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Primary transcripts containing miRNAMost miRNAs are transcribed by RNA polymerase II from noncoding DNA regions that generate short dsRNA hairpinsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Fig. 16.25amiRNA processing Drosha excises stem-loop from primary miRNA (pri-miRNA) to generate pre-miRNA of ~ 70 ntDicer processes pre-miRNA to a mature duplex miRNAOne strand is incorporated into miRNA-induced silencing complex (RISC) Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Fig. 16.26Two ways that miRNAs can down-regulate expression of target genesWhen complementarity is perfect:Target mRNA is degradedCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*When complementarity is imperfect: Translation of mRNA target is repressedFig. 16.27siRNAs detect and destroy foreign dsRNAsTwo biological sources of dsRNAs that are precursors of siRNAs (pri-RNAs) Transcription of both strands of an endogenous genomic sequenceArise from exogenous viruspri-RNAs are processed by DicersiRNA pathway targets dsRNAs for degradationsiRNAs are very useful experimental tools to selectively knock down expression of target genesTo study function of a gene, dsRNAs for that gene can be introduced into cellsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Posttranslational modifications of proteinsUbiquitination – covalent attachment of ubiquitin to other proteins targets those proteins for degradation by the proteosomeCascades of phosphorylation and dephosphorylationTransmission of signals across the cell membrane to the nucleus (discussed more in Chapter 17)Sensitization – tissues exposed to hormones for long periods of time lose ability to respond to the hormoneExample: binding of epinephrine to β-adrenergic receptors on surface of heart muscle cells (see Fig 16.28)Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Phosphorylation and desensitization of β-adrenergic receptor Phosphorylation of receptor has no effect on its binding to epinephrine, but blocks its downstream functionsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Fig. 16.28Computer analyses can reveal regulatory mechanismsCan translate open reading frames into in silico proteins Recognize motifs within proteins e.g. Zinc-finger domains or sites for posttranslational modificationPhlyogenetic footprinting – compare noncoding DNA sequences of closely related speciesSequences that are conserved may have important functions in gene regulationChIP technology – see Chapter 10Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*A comprehensive example of gene regulation: Sex determination in DrosophilaIn Drosophila, ratio of X chromosomes to autosomal chromosomes (X:A ratio) determines sex, fertility, and viabilityX:A ratio influences sex through three independent pathways:Male vs female appearance and behaviorDevelopment of germ cells as eggs or spermDosage compensation – males have increased transcription of X-linked genesCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Sex-specific traits in DrosophilaCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Fig. 16.29How chromosomal constitution affectsphenotype in DrosophilaCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Table 16.2Drosophila mutations that affect the two sexes differently*Sxfl is a recessive mutation of Sex lethal**SxlML is a dominant mutation of Sex lethalCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Table 16.3The X:A ratio regulates transcription of the Sxl geneSeveral HLH transcription factors are key regulators of sex determinationX-linked genes are "numerator elements"Autosomal genes are "denominator elements"Numerator/denominator determines ratio of HLH homodimers and heterodimersOnly the homodimer activates transcription of Sxl Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Fig. 16.30The Sxl protein triggers a cascade of splicing of transformer (tra) and double-sex (dsx) mRNAsCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Fig. 16.31Sxl protein regulates splicing of tra mRNATra protein regulates splicing of dsx mRNADsx-F is a transcriptional activator and Dsx-M is a transcriptional repressorCopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Fig. 16.32The Tra protein regulates splicing of fruitless (fru) mRNACopyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al., 4th edition, Chapter 16*Fig. 16.33