Benjamin Lewin - GBV · 2007. 10. 7. · The consequences of modification and restriction 506 ... Spm elements influence gene expression 588 The role of transposable elements in hybrid

Benjamin Lewin

Jechnische Hochschule DarmstadtFACKBEREiCH 10 - BIOLOGIE

- B i b l i o t h e k -Schnittspahnstrafle 10

0-64287 DarmsUAt,

Inv.-Nr.

Oxford New York TokyoOxford University Press

1997

Outline

Introduction: Cells as macromolecularassemblies 1

1 Proteins 32 Compartments 27

PART 1: DNA as information 493 Genes are mutable units 514 DNA is the genetic material 715 Nucleic acid structure 976 Isolating the gene 115

PART 2: From gene to protein 1517 Messenger RNA 1538 Protein synthesis 1799 Interpreting the genetic code 213

10 Protein localization 244

PART 3: Prokaryotic geneexpression 285

11 Transcription 28712 The operon 33513 Phage strategies 395

PART 4: Perpetuation of DNA 42714 The replicon 42915 DNA replication 47116 Restriction and repair 50517 Recombination 53118 Transposons 56319 Retroviruses and retroposons 597

PART 5: The eukaryoticgenome 621

20 DNA biotechnology 62321 Genomes 64522 Exons and introns 66323 Gene numbers 68724 Organelle genomes 71325 Simple sequence DNA 72726 Chromosomes 74327 Nucleosomes 769

PART 6: Eukaryotic geneexpression 809

28 Initiation of transcription 81129 Regulation of transcription 84730 Nuclear splicing 88531 Catalytic RNA 92132 Rearrangement of DNA 94733 Immune diversity 989

PART 7: Cell growth, cancer, anddevelopment 1025

34 Protein trafficking 102735 Signal transduction 105336 Cell cycle and growth regulation 108937 Oncogenes and cancer 113138 Gradients and cascades 1173

Epilogue: Landmark shifts inperspectives 1213

Contents

Introduction: Cells as macromolecular assemblies

1: ProteinsMacromolecules are assembled by polymerizing small moleculesProteins consist of chains of amino acidsProtein conformation depends on the aqueous environmentProtein structures are extremely versatileHow do proteins fold into the correct conformation?

2: CompartmentsCellular compartments are bounded by membranesThe cytoplasm contains networks of membranesCell shape is determined by the cytoskeletonSome organelles are surrounded by an envelopeThe environment of the nucleus and its reorganizationThe role of chromosomes in heredity

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Part 1: DNA as information

3: Genes are mutable unitsDiscovery of the geneGenes lie in a linear array on chromosomesOne gene—one proteinThe cistronMapping mutations at the molecular levelThe nature of multiple alleles

4: DNA is the genetic materialThe discovery of DNADNA is the (almost) universal genetic materialThe components of DNADNA is a double helix

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DNA replication is semiconservativeThe genetic code is read in tripletsMutations change the sequence of DNAMutations are concentrated at hotspotsThe rate of mutation

5: Nucleic acid structureDNA can be denatured and renaturedNucleic acids hybridize by base pairingSingle-stranded nucleic acids may have secondary structureInverted repeats and secondary structureDuplex DNA has alternative double-helical structuresClosed DNA can be supercoiledSupercoiling influences the structure of the double helix

6: Isolating the geneA restriction map is constructed by cleaving DNA into specific fragmentsRestriction sites can be used as genetic markersObtaining the sequence of DNAProkaryotic genes and proteins are colinearm-acting sites and trans-acting moleculesEukaryotic genes are often interruptedSome DNA sequences code for more than one proteinGenetic information can be provided by DNA or RNAThe scope of the paradigm

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Part 2: From gene to protein

7: Messenger RNATransfer RNA is the adaptorMessenger RNA is translated by ribosomesThe life cycle of messenger RNAMost bacterial genes are expressed via polycistronic messengersTranslation of eukaryotic mRNAEukaryotic mRNAs are polyadenylated at the 3' endEukaryotic mRNAs have a methylated cap at the 5' endProcessing and stability of mRNA

8: Protein synthesisOrganization of the ribosomeThe stages of protein synthesisInitiation in bacteria needs 30S subunits and accessory factorsA special initiator tRNA starts the polypeptide chainInitiation involves base pairing between mRNA and rRNASmall subunits migrate to initiation sites on eukaryotic mRNAElongation factor T brings aminoacyl-tRNA into the A site

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Contents I xi

Translocation moves the ribosomeThree codons terminate protein synthesisRibosomes have several active centersThe role of ribosomal RNA in protein synthesis

9: Interpreting the genetic codeCodon-anticodon recognition involves wobblingtRNA contains modified bases that influence its pairing propertiesThe genetic code is altered in mitochondriatRNAs are charged with amino acids by individual synthetasesAccuracy depends on proofreadingSuppressor tRNAs have mutated anticodons that read new codonsThe accuracy of translationtRNA may influence the reading frame

10: Protein localizationChaperones may be required for protein foldingPost-translational membrane insertion depends on leader sequencesA hierarchy of sequences determines location within organellesSignal sequences initiate co-translational transfer through ER membranesHow do proteins enter and leave membranes?The translocation apparatus interacts with signal and anchor sequencesAnchor signals are needed for membrane residenceBacteria use both co-translational and post-translational translocationPores control nuclear ingress and egressProtein degradation by proteasomes

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Part 3: Prokaryotic gene expression 285

11: TranscriptionTranscription is catalyzed by RNA polymeraseRNA polymerase consists of multiple subunitsSigma factor controls binding to DNAPromoter recognition depends on consensus sequencesRNA polymerase binds to one face of DNASubstitution of sigma factors may control initiationSporulation utilizes a cascade of many sigma factorsBacterial RNA polymerase has two modes of terminationHow does rho factor work?Antitermination depends on specific sitesMore subunits for RNA polymerase

12: The operonStructural gene clusters are coordinately controlledRepressor is controlled by a small molecule inducerMutations identify the operator and the regulator gene

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Repressor protein binds to the operator and is released by inducerThe specificity of protein-DNA interactionsRepression can occur at multiple lociDistinguishing positive and negative controlCatabolite repression involves positive regulation at the promoterAdverse growth conditions provoke the stringent responseAutogenous control may occur at translationAlternative secondary structures control attenuationSmall RNA molecules can regulate translationRegulation by cleavage of mRNACleavages are needed to release prokaryotic and eukaryotic rRNAs

13: Phage strategiesLytic development is controlled by a cascadeFunctional clustering in phages T7 and T4The lambda lytic cascade relies on antiterminationLysogeny is maintained by an autogenous circuitThe DNA-binding form of repressor is a dimerRepressor binds cooperatively at each operator using a helix-turn-helix motifHow is repressor synthesis established?A second repressor is needed for lytic infectionA delicate balance: lysogeny versus lysis

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Part 4: Perpetuation of DNA

14: The repliconOrigins can be mapped by autoradiography and electrophoresisThe bacterial genome is a single circular repliconEach eukaryotic chromosome contains many repliconsIsolating the origins of yeast repliconsD loops may be maintained at mitochondrial originsThe problem of linear repliconsRolling circles produce multimers of a repliconSingle-stranded genomes are generated for bacterial conjugationConnecting bacterial replication to the cell cycleCell division and chromosome segregationMultiple systems ensure plasmid survival in bacterial populationsPlasmid incompatibility is connected with copy number

15: DNA replicationDNA polymerases: the enzymes that make DNADNA synthesis is semidiscontinuous and primed by RNAThe primosome initiates synthesis of Okazaki fragmentsCoordinating synthesis of the lagging and leading strandsThe replication apparatus of phage T4Creating the replication forks at an origin

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Contents I xiii

Common events in priming replication at the origin 496Does methylation at the origin regulate initiation? 498Licensing factor controls eukaryotic rereplication 500

16: Restriction and repair 505The consequences of modification and restriction 506Type II restriction enzymes are common 508The alternative activities of type I enzymes " 510The dual activities of type III enzymes 513Dealing with injuries in DNA 515Excision repair systems in E. coli 518Controlling the direction of mismatch repair 521Retrieval systems in E. coli 523RecA triggers the SOS system 525Eukaryotic repair systems 527

17: Recombination 531Breakage and reunion involves heteroduplex DNA 534Double-strand breaks initiate recombination 537Double-strand breaks may initiate synapsis 539Bacterial recombination involves single-strand assimilation 542Gene conversion accounts for interallelic recombination 548Topological manipulation of DNA 550Gyrase introduces negative supercoils in DNA 553Specialized recombination involves breakage and reunion at specific sites 555

18: Transposons 563Insertion sequences are simple transposition modules 565Composite transposons have IS modules 567Transposition occurs by both replicative and nonreplicative mechanisms 569Common intermediates for transposition . 572Replicative transposition proceeds through a cointegrate 574Nonreplicative transposition proceeds by breakage and reunion 576TnA transposition requires transposase and resolvase 578Transposition of TnlO has multiple controls 580Controlling elements in maize cause breakage and rearrangements 583Controlling elements in maize form families of transposons 586Spm elements influence gene expression 588The role of transposable elements in hybrid dysgenesis 589

19: Retroviruses and retroposons 597The retrovirus life cycle involves transposition-like events 598Retroviruses may transduce cellular sequences 607Yeast Ty elements resemble retroviruses 609Many transposable elements reside in D. melanogaster 611Retroposons fall into two classes 613

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Part 5: The eukaryotic genome 621

20: DNA biotechnologyAny DNA sequence can be cloned in bacteria or yeastConstructing the chimeric DNACopying mRNA into cDNAIsolating individual genes from the genomeWalking along the chromosomeEukaryotic genes can be expressed in prokaryotic systems

21: GenomesThe C-value paradox describes variations in genome sizeReassociation kinetics depend on sequence complexityEukaryotic genomes contain several sequence componentsNonrepetitive DNA complexity can estimate genome sizeEukaryotic genomes contain repetitive sequencesMost structural genes lie in nonrepetitive DNAHow many nonrepetitive genes are expressed?Genes are expressed at widely varying levels

22: Exons and introns ,Organization of interrupted genes may be conservedGenes show a wide distribution of sizesOne DNA sequence may code for multiple proteinsExon sequences are conserved but introns varyGenes can be isolated by the conservation of exonsHow did interrupted genes evolve?

23: Gene numbersEssential genes and total gene numberGlobin genes are organized in two clustersUnequal crossing-over rearranges gene clustersGene clusters suffer continual reorganizationSequence divergence is the basis for the evolutionary clockPseudogenes are dead ends of evolutionGenes for rRNA comprise a repeated tandem unitAn evolutionary dilemma: how are multiple active copies maintained?

24: Organelle genomesOrganelle genomes are circular DNAs that code for organelle proteinsThe chloroplast genome codes for -100 proteins and RNAsThe mitochondrial genome is large in yeast but small in mammalsRecombination and rearrangement of organelle DNA

25: Simple sequence DNASatellite DNAs often lie in heterochromatinArthropod satellites have very short identical repeats

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Mammalian satellites consist of hierarchical repeatsEvolution of hierarchical variations in the satelliteThe consequences of unequal crossing-overCrossover fixation could maintain identical repeatsMinisatellites are useful for genetic mapping

26: ChromosomesCondensing viral genomes into their coatsThe bacterial genome is a nucleoid with many supercoiled loopsLoops, domains, and scaffolds in eukaryotic DNAThe contrast between interphase chromatin and mitotic chromosomesThe extended state of lampbrush chromosomesTranscription disrupts the structure of polytene chromosomesThe eukaryotic chromosome as a segregation deviceTelomeres seal the ends of chromosomes

27: NucleosomesThe nucleosome is the subunit of all chromatinDNA is coiled in arrays of nucleosomesDNA structure varies on the nucleosomal surfaceSupercoiling and the periodicity of DNAThe path of nucleosomes in the chromatin fiberOrganization of the histone octamerReproduction of chromatin requires assembly of nucleosomesDo nucleosomes lie at specific positions?Are transcribed genes organized in nucleosomes?DNAase hypersensitive sites change chromatin structureDomains define regions that contain active genesHeterochromatin is created by interactions with histones

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Part 6: Eukaryotic gene expression 809

28: Initiation of transcriptionEukaryotic RNA polymerases consist of many subunitsPromoter elements are defined by mutations and footprintingRNA polymerase I has a bipartite promoterRNA polymerase III uses both downstream and upstream promotersThe basal apparatus consists of RNA polymerase II and general factorsA connection between transcription and repairPromoters for RNA polymerase II have short sequence elementsEnhancers contain bidirectional elements that assist initiationIndependent domains bind DNA and activate transcriptionInteraction of upstream factors with the basal apparatus

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29: Regulation of transcription 847Response elements identify genes under common regulation 848There are many types of DNA-binding domains 850A zinc finger motif is a DNA-binding domain 852Steroid receptors have several independent domains 855Homeodomains bind related targets in DNA 859Helix-loop-helix proteins interact by combinatorial association 862Leucine zippers are involved in dimer formation 864Dynamic versus pre-emptive models for gene activation 866Long range regulation and insulation of domains 871Gene expression is associated with demethylation 875Methylation is responsible for imprinting 878

30: Nuclear splicing 885Nuclear splice junctions are interchangeable but are read in pairs 887Nuclear splicing proceeds through a lariat 891SnRNAs are required for splicing and form a spliceosome 893Group II introns autosplice via lariat formation 901Alternative splicing involves differential use of splice junctions 904eis-splicing and Jrans-splicing reactions 907Yeast tRNA splicing involves cutting and rejoining 9113' ends are generated by termination and by cleavage reactions 913

31: Catalytic RNA 921Group I introns undertake self-splicing by transesterification 922Group I introns form a characteristic secondary structure 926Ribozymes have various catalytic activities 928Some introns code for proteins that sponsor mobility 931RNA can have ribonuclease activities . 935RNA editing utilizes information from several sources 937

32: Rearrangement of DNA 947The mating pathway is triggered by signal transduction 948Yeast can switch silent and active loci for mating type 952Silent cassettes at HML and HMR are repressed 956Unidirectional transposition is initiated by the recipient MAT locus 958Regulation of HO expression 960Trypanosomes rearrange DNA to express new surface antigens 962Interaction of Ti plasmid DNA with the plant genome 967Selection of amplified genomic sequences 975Exogenous sequences can be introduced into cells and animals by transfection 979

33: Immune diversity 989Clonal selection amplifies lymphocytes that respond to individual antigens 992Immunoglobulin genes are assembled from their parts in lymphocytes 994The diversity of germline information 1000Recombination between V and C genes generates deletions and rearrangements 1002Allelic exclusion is triggered by productive, rearrangement 1007DNA recombination causes class switching 1009Early heavy chain expression can be changed by RNA processing 1011

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Somatic mutation generates additional diversityT-cell receptors are related to immunoglobulinsThe major histocompatibility locus codes for many genes of the immune system

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Part 7: Cell growth, cancer, and development 102b

34: Protein traffickingOligosaccharides are added to proteins in the ER and GolgiCoated vesicles transport both exported and imported proteinsProtein localization depends on further signalsReceptors recycle via endocytosis

35: Signal transductionCarriers and channels form water-soluble paths through the membraneG proteins may activate or inhibit target proteinsProtein tyrosine kinases induce phosphorylation cascadesThe Ras pathwayActivating MAP kinase pathwaysCyclic AMP and activation of CREBThe JAR-STAT pathway

36: Cell cycle and growth regulationCycle progression depends on discrete control pointsM phase kinase is a dimer that regulates entry into mitosisProtein phosphorylation and dephosphorylation control the cell cyclep34 (cdc2 or CDC28) is the key regulator in yeastsCDC28 acts at both START and mitosis in 5. cerevisiaeMany cdk-cyclin complexes are found in animal cellsFunctions of cdc2-cyclin and cdk-cyclin dimersG0/G1 and Gl/S transitions involve cdk inhibitorsReorganization of the cell at mitosisApoptosis

37: Oncogenes and cancerTransforming viruses carry oncogenesRetroviral oncogenes have cellular counterpartsRas proto-oncogenes can be activated by mutationInsertion, translocation, or amplification may activate proto-oncogenesOncogenes code for components of signal transduction cascadesGrowth factor receptor kinases and cytoplasmic tyrosine kinasesOncoproteins may regulate gene expressionRB is a tumor suppressor that controls the cell cycleThe tumor suppressor p53 suppresses growth or triggers apoptosisImmortalization and transformation

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38: Gradients and cascades 1173A gradient must be converted into discrete compartments 1175Maternal gene products establish gradients in early embryogenesis 1177Anterior-posterior development uses localized gene regulators 1180Dorsal-ventral development uses localized receptor-Iigand interactions 1184Cell fate is determined by compartments that form by the blastoderm stage 1191Complex loci are extremely large and involved in regulation 1198The homeobox is a common coding motif in homeotic genes 1205

Epilogue: Landmark shifts in perspectives 1213

Glossary 1217

Index 1241