ENOME 5T.A. BROWN

Abbreviations

xxi

Preface

xxii i

Acknowledgements

xxv

An Introduction to GENOMES

xxvi i

PART

i

How Genomes are Studied

xxx

Chapter I What is a Genome?

11 .1

The Human Genome

31 .1 .1 The physical structure of the human genome

3

1 .1 .2 The genetic content of the human genome

6Box 1 .1 Some key facts about genes

71 .2

Genomes of Other Organisms

7

1 .2 .1 Genomes of eukaryotes

81 .2 .2 Genomes of prokaryotes

91 .3

Why are Genome Projects Important?

1 1

Chapter 2 Mapping Genomes by Genetic Techniques

1 3

2 .1

Genetic and Physical Maps

15

2 .2

Markers for Genetic Maps

1 5

2.2.1 Genes were the first markers to be used

1 5Box 2.1 A quick guide to Mendelian genetics

1 72 .2 .2 DNA markers for genetic mapping

1 8Restriction fragment length polymorphisms (RFLPs)

1 8Simple sequence length polymorphisms (SSLPs)

1 8Single nucleotide polymorphisms (SNPs)

212 .3

Approaches to Genetic Mapping

2 22.3.1 Linkage analysis is the basis of genetic mapping

2 2The behavior of chromosomes during meiosis explains why genes display

partial and not complete linkage

2 2From partial linkage to genetic mapping

2 52.3 .2 Carrying out linkage analysis with different types of organism

26Linkage analysis when planned breeding experiments are possible

27Gene mapping by human pedigree analysis

29

fox 2.2 Multipoint crosses

3 0Genetic mapping in bacteria

32

Chapter 3 Mapping Genomes by Physical Techniques

3 7

3 .1

Restriction Mapping

393 .1 .1 The basic methodology for restriction mapping

39

3 .1 .2 The scale of restriction mapping is limited by the sizes of the restriction fragments

4 1Restriction mapping with rare cutters

41Monitoring the results of restriction with a rare cutter

44

3.2

FISH - Fluorescent In Situ Hybridization

453.2 .1 In situ hybridization with radioactive or fluorescent probes

453 .2.2 FISH in action

47Low-resolution FISH with metaphase chromosomes

47High-resolution FISH with extended chromosomes and DNA fibers

473 .3

Sequence Tagged Site (STS) Mapping

483 .3 .1 Any unique DNA sequence can be used as an STS

493 .3.2 Fragments of DNA for STS mapping

49Radiation hybrids

50A clone library can also be used as the mapping reagent for STS analysis

513.4

Progress Towards the Human Genome Map

54

Chapter 4 Sequencing Genomes

594.1

The Methodology for DNA Sequencing

604.1.1 Chain termination DNA sequencing

60Chain termination sequencing in outline

6 1Box 4.1 The chemical degradation sequencing method

6 1Chain termination sequencing requires a single-stranded DNA template

64Box 4.2 DNA polymerases for chain termination sequencing

66The primer determines the region of the template DNA that will be sequenced

67Thermal cycle sequencing offers an alternative to the traditional methodology

67Fluorescent primers are the basis of automated sequence reading

6 84 .1 .2 Radical departures from conventional DNA sequencing

6 94 .2

Assembly of a Contiguous DNA Sequence

7 04 .2 .1 Sequence assembly by the shotgun approach

7 0The potential of the shotgun approach was proven by the Haemophilus

influenzae sequence

70The strengths and limitations of the shotgun approach

734.2.2 Sequence assembly by the clone contig approach

73Cloning vectors for large pieces of DNA

73Clone contigs can be built up by chromosome walking, but the method is laborious

75Newer, more rapid methods for clone contig assembly

784 .2.3 The directed shotgun approach

794 .3

The Sequencing Phase of the Human Genome Project

8 04.3.1 The Human Genome Project is now largely based on BAC libraries

8 14 .3 .2 Rapid sequence acquisition can be directed at interesting regions of the genome

8 1EST sequencing is a rapid way of accessing genes

8 1Sequence skimming is equally rapid

8 14 .3 .3 The sequencing phase of the Human Genome Project raises ethical questions

82

Chapter 5 Understanding a Genome Sequence

855.1

Locating Genes in DNA Sequences

865 .1 .1 Gene location by sequence inspection

ß6Genes are open reading frames

ß6Simple ORF scans are less effective with higher eukaryotic DNA

87Homology searches give an extra dimension to sequence inspection

8 95 .1 .2 Experimental techniques for gene location

8 9Hybridization tests can determine if a fragment contains expressed sequences

8 9cDNA sequencing enables genes to be mapped within DNA fragments

9 0Methods are available for precise mapping of the ends of transcripts

9 1Exon-intron boundaries can also be located with precision

9 25 .2

Determining the Function of a Gene

925.2.1 Computer analysis of gene function

93Homology reflects evolutionary relationships

93

Homology analysis can provide information on the function of an entire gene or ofsegments within it

94Homology analysis in the yeast genome project

955 .2 .2 Assigning gene function by experimental analysis

96Gene inactivation is the key to functional analysis

96Methods for inactivating specific genes depend on homologous recombination

97The phenotypic effect of gene inactivation is sometimes difficult to discern

98Gene overexpression can also be used to assess function

985 .2 .3 More detailed studies of the activity of a protein coded by an unknown gene

100Directed mutagenesis can be used to probe gene function in detail

100Reporter genes and immunocytochemistry can be used to locate where and when

genes are expressed

102Protein-protein interactions can be examined by phage display and the yeas t

two-hybrid system

1035.3

Comparative Genomics

1035 .3 .1 Comparative genomics as an aid to gene mapping

1035 .3 .2 Comparative genomics in the study of human disease genes

1065.4

From Genome to Cell

1065 .4 .1 Patterns of gene expression

107Gene expression is assayed by measuring levels of RNA transcripts

107The proteome

109

- PAR Tf

g

How Genomes Function

11 2Chapter 6 Genome Anatomies

11 56 .1

The Anatomy of the Eukaryotic Genome

1166 .1 .1 Eukaryotic nuclear genomes

117Packaging of DNA into chromosomes

117The special features of metaphase chromosomes

118Where are the genes in a genome?

119Box 6.1 Unusual chromosome types

120Families of genes

120Box 6.2 How many genes in the human genome?

12 2Box 6.3 Two examples of unusual gene organization

124Pseudogenes and other gene relics

1246 .1 .2 Eukaryotic organelle genomes

125Physical features of organelle genomes

125The genetic content of organelle genomes

125The origins of organelle genomes

1266 .2

The Anatomy of the Prokaryotic Genome

1286 .2 .1 The physical structure of the prokaryotic genome

128The traditional view of the bacterial 'chromosome'

12 9Complications on the E. coli theme

12 96 .2 .2 The genetic organization of the prokaryotic genome

132Operons are characteristic features of prokaryotic genomes

133Speculations regarding the content of the minimal genome and the identity of

distinctiveness genes

1346 .3

The Repetitive DNA Content of Genomes

134Box 6.4 The organization of the human genome

13 56 .3 .1 Tandemly repeated DNA

13 6Satellite DNA is found at centromeres and elsewhere in eukaryotic chromosomes

13 6Mini- and microsatellites

13 66 .3 .2 Interspersed genome-wide repeats

137

Transposition via an RNA intermediate

137DNA transposons

1396.3 .3 Repetitive DNA and eukaryotic chromosome architecture

140

Chapter 7 The Role of DNA-binding Proteins

1437.1

The Structure of DNA

1447.1 .1 Nucleotides and polynucleotides

145The chemical structures of the four nucleotides

145Polynucleotides

145RNA

1457.1 .2 The double helix

14 5The key features of the double helix

147The double helix has structural flexibility

147Box 7 .1 Base-pairing in RNA

477.2

Proteins

1507.2 .1 The four levels of protein structure

15 0Amino acid diversity underlies protein diversity

15 3Box 7 .2 Noncovalent bonds in proteins

5 3Primary structure dictates protein function

1547.3

Methods for Studying DNA-binding Proteins

15 67.3 .1 Locating a protein-binding site on a DNA molecule

15 6Gel retardation identifies DNA fragments that bind to proteins

15 6Protection assays pinpoint binding sites with greater accuracy

15 7Modification interference identifies nucleotides central to protein binding

15 77 .3 .2 Purification of a DNA-binding protein

15 77.3 .3 Techniques for studying protein and DNA-protein structures

16 0X-ray crystallography has broad applications in structure determination

160NMR gives detailed structural information for small proteins

16 07 .4

Interactions Between DNA and DNA-binding Proteins

1627 .4 .1 The DNA partner in the interaction

163Direct readout of the nucleotide sequence

163The nucleotide sequence has a number of indirect effects on helix structure

1637 .4 .2 The protein partner

164The helix-turn-helix motif is present in prokaryotic and eukaryotic proteins

164Box 7.3 RNA-binding motifs

65

Zinc fingers are common in eukaryotic proteins

166Other DNA-binding motifs

16 77 .4 .3 DNA and protein in partnership

16 7Contacts between DNA and proteins

167Can sequence specificity be predicted from the structure of a recognition helix?

168

Chapter 8 Initiation of Transcription: the First Step in Gene Expression

17 18 .1

Accessing the Genome

1738 .1 .1 Effects of chromatin packaging on eukaryotic gene expression

173Heterochromatin, euchromatin and chromatin loops

173Structural and functional domains

174Nucleosome positioning is important in modulating gene expression

175Box 8J DNA methylation and gene expression

778 .2

Assembly of the Transcription Initiation Complexes of Prokaryotes and Eukaryotes

1788 .2 .1 RNA polymerases

178Box 8.2 Mitochondria! and chloroplast RNA polymerases

1788 .2 .2 Recognition sequences for transcription initiation

179Bacterial RNA polymerases bind to promoter sequences

179Eukaryotic promoters are more complex

1798 .2 .3 Assembly of the transcription initiation complex

181

Transcription initiation in E . coli

181Box 8 .3 Initiation of transcription in the archaea

18 ITranscription initiation with RNA polymerase II

182Transcription initiation with RNA polymerases I and III

1828 .3

Regulation of Transcription Initiation

1858 .3 .1 Strategies for controlling transcription initiation in bacteria

185Promoter structure determines the basal level of transcription initiation

185Regulatory control over bacterial transcription initiation

186- ox 8.4 Cis and trans

18 88 .3 .2 Control of transcription initiation in eukaryotes

188Activators of eukaryotic transcription initiation

189Box 8 .5 Regulation of NA polymerase 1 initiation

18 9Contacts between transcription factors and the preinitiation complex

190A few repressors of eukaryotic transcription initiation have been identified

190Control over the activity of transcription factors

19 1

Chapter 9 Synthesis and Processing of RNA

I959 .1

The RNA Content of a Cell

1969 .1 .1 Coding and noncoding RNA

1969 .1 .2 Precursor RNA

1979 .1 .3 RNA in time and space

198The population of RNAs in a cell changes over time

198In eukaryotes, RNAs must be transported from nucleus to cytoplasm, and

possibly back again

1999 .2

Synthesis and Processing of mRNAs

2019 .2 .1 Synthesis of bacterial mRNAs

201The elongation phase of bacterial transcription

201Termination of bacterial transcription

203Control over the choice between elongation and termination

203Box 9 .1 Antitermination during the infection cycle of bacteriophage

2079 .2 .2 Synthesis of eukaryotic mRNAs by RNA polymerase II

207Capping of RNA polymerase II transcripts occurs immediately after initiation

207Elongation of eukaryotic mRNAs

209Termination of mRNA synthesis is combined with polyadenylation

2109 .2 .3 Intron splicing

212Conserved sequence motifs indicate the key sites in GU-AG introns

213Box 9 .2 Processed pseudogenes

21 4Outline of the splicing pathway for GU-AG introns

214snRNAs and their associated proteins are the central components of the splicin g

apparatus

215SR proteins are mediators in splice site selection

216AU-AC introns are similar to GU-AG introns but require a different splicin g

apparatus

2169.3

Synthesis and Processing of Noncoding RNAs

2199 .3 .1 Transcript elongation and termination by RNA polymerases I and III

2199 .3 .2 Cutting events involved in processing of bacterial and eukaryotic pre-rRNA an d

pre-tRNA

2199 .3 .3 Introns in eukaryotic pre-rRNA and pre-tRNA

221Eukaryotic pre-rRNA introns are self-splicing

221Eukaryotic tRNA introns are variable but all are spliced by the same mechanism

2229.4

Processing of Pre-RNA by Chemical Modification

222Box 9 .3 Other types of intron

2259 .4 .1 Chemical modification of pre-rRNAs

225Box 9 .4 Chemical modification without snoRNAs

2269 .4 .2 RNA editing

226

9.5

Turnover of mRNAs

227Box 9 .5 More complex forms of RNA editing

228

Chapter I 0 Synthesis and Processing of the Proteome

23 I10 .1

The Role of tRNA in Protein Synthesis

23 210 .1 .1 Aminoacylation: the attachment of amino acids to tRNAs

23 2All tRNAs have a similar structure

23 3Aminoacyl-tRNA synthetases attach amino acids to tRNAs

23410 .1 .2 Codon-anticodon interactions : the attachment of tRNAs to mRNA

235The genetic code specifies how an mRNA sequence is translated into a polypeptide

235Codon-anticodon recognition is made flexible by wobble

238Box 10.1 The origin and evolution of the genetic code

24010.2

The Role of the Ribosome in Protein Synthesis

240Box 10.2 The compactness of vertebrate mitochondria) genomes

24210.2 .1 Ribosome structure

242Ultracentrifugation was used to measure the sizes of ribosomes and thei r

components

242Probing the fine structure of the ribosome

24210.2 .2 Translation in detail

243Initiation in bacteria requires an internal ribosome binding site

243Initiation in eukaryotes is mediated by the cap structure and poly(A) tail

245Elongation is similar in bacteria and eukaryotes

246Box I0.3 Initiation of eukaryotic translation without scanning

247Box 10.4 Regulation of translation initiation

248Termination requires release factors

24810 .3

Post-translational Processing of Proteins

248Box 10.5 Frameshifting

25010 .3 .1 Protein folding

250Not all proteins fold spontaneously in the test tube

250Box 10.6 Translation in the archaea

25 1In cells, folding is aided by molecular chaperones

25210 .3 .2 Processing by proteolytic cleavage

25 3Cleavage of the ends of polypeptides

25 3Proteolytic processing of polyproteins

25 410 .3.3 Processing by chemical modification

25 510 .3.4 Inteins

25 610 .4

Protein Turnover

25 810 .4.1 Degradation of ubiquitin-tagged proteins in the proteasome

25 9

Chapter I I Regulation of Genome Activity

26311 .1

Transient Changes in Genome Activity

26611 .1 .1 Signal transmission by import of the extracellular signaling compound

267Lactoferrin is an extracellular signaling compound which acts as a transcriptio n

factor

267Some imported signaling compounds directly influence the activity o f

pre-existing transcription factors

268Some imported signaling compounds influence genome activity indirectly

26811 .1 .2 Signal transmission mediated by cell surface receptors

270The simplest signal transduction pathways involve direct activation o f

transcription factors

270More complex signal transduction pathways have many steps between receptor

and genome

273Signal transduction via second messengers

27511 .2

Permanent and Semipermanent Changes in Genome Activity

27711 .2 .1 Genome rearrangements

278

Yeast mating types are determined by gene conversion events

278Genome rearrangements are responsible for immunoglobulin and T-cell receptor

diversities

27811 .2 .2 Changes in chromatin structure

28011 .2 .3 Genome regulation by feedback loops

28111 .3

Regulation of Genome Activity During Development

28211 .3 .1 Sporulation in Bacillus

282Sporulation involves coordinated activities in two distinct cell types

282Special u subunits control genome activity during sporulation

28411 .3 .2 Vulval development in Caenorhabditis elegans

285C . elegans is a model for multicellular eukaryotic development

285Determination of cell fate during development of the C . elegans vulva

28611 .3 .3 Development in Drosophila melanogaster

287Maternal genes establish protein gradients in the Drosophila embryo

289A cascade of gene expression converts positional information into a

segmentation pattern

289Segment identity is determined by the homeotic selector genes

291Homeotic selector genes are universal features of higher eukaryotic development

292Box 11 .1 The genetic basis of flower development

292

3 . ► > PART 9

How Genomes Replicate and Evolve

296

Chapter 12 Genome Replication

29912 .1

The Issues Relevant to Genome Replication

30 012 .2

The Topological Problem

30 112 .2.1 Experimental proof of the Watson-Crick scheme for DNA replication

30 1The Meseison-Stahl experiment

30 2Box 12.1 Variations on the semiconservative theme

30312 .2 .2 DNA topoisomerases provide a solution to the topological problem

30 512 .3

The Replication Process

306Box 12.2 The diverse functions of DNA topoisomerases

30612 .3 .1 Initiation of genome replication

307Initiation at the E. coli origin of replication

307Origins of replication in yeast have also been clearly defined

308Replication origins in higher eukaryotes have been less easy to identify

30912 .3 .2 The elongation phase of replication

311The DNA polymerases of bacteria and eukaryotes

311Discontinuous strand synthesis and the priming problem

314Events at the bacterial replication fork

314The eukaryotic replication fork: variations on the bacterial theme

317Box 12.3 Genome replication in the archaea

31 812 .3 .3 Termination of replication

319Replication of the E. coli genome terminates within a defined region

319Little is known about termination of replication in eukaryotes

321Box 12.4 Telomeres in Drosophila

32212 .3 .4 Maintaining the ends of a linear DNA molecule

322Telomeric DNA is synthesized by the telomerase enzyme

32212 .4

Regulation of Eukaryotic Genome Replication

32312 .4.1 Coordination of genome replication and cell division

323Establishment of the pre-replication complex enables genome replication t o

commence

325Regulation of pre-RC assembly

32612 .4 .2 Control within S phase

326

Early and late replication origins

326Checkpoints within S phase

326

Chapter 13 The Molecular Basis of Genome Evolution

32913 .1

Mutations

33 113 .1 .1 The causes of mutations

331Box 13 .1 Terminology for describing point mutations

332Errors in replication are a source of point mutations

332Replication errors can also lead to insertion and deletion mutations

335Mutations are also caused by chemical and physical mutagens

337

13 .1 .2 The effects of mutations

340The effects of mutations on genomes

340The effects of mutations on multicellular organisms

343The effects of mutations on microorganisms

34413 .1 .3 Hypermutation and the possibility of programmed mutations

34513 .1 .4 DNA repair

346Box 13.2 DNA repair and human disease

348Direct repair systems fill in nicks and correct some types of nucleotide

modification '

348Base excision repairs many types of damaged nucleotide

348:ox 13.3 Cell cycle checkpoints for monitoring DNA damage

349Nucleotide excision repair is used to correct more extensive types of damage

349Mismatch repair: correcting errors of replication

35113 .2

Recombination

35313 .2 .1 Homologous recombination

35 3The Holliday model for homologous recombination

353Proteins involved in homologous recombination in E. coli

355The double-strand break model for recombination in yeast

35 6Box 13 .4 Double-strand break repair

35 713 .2.2 Site-specific recombination

357Integration of A, DNA into the E. coli genome involves site-specifi c

recombination

357Box 13 .5 The RecE and RecF recombination pathways of E. coil

35 913 .2.3 Transposition

359Replicative and conservative transposition of DNA transposons

35 9Tranposition of retroelements

36 3

Chapter 14 Patterns of Genome Evolution

36 714 .1

Genomes: The First Ten Billion Years

36814 .1 .1 The origins of genomes

36 9The first biochemical systems were centered on RNA

369The first DNA genomes

370Box 14.1 How unique is life?

37 114 .2

Acquisition of New Genes

37214 .2.1 Acquisition of new genes by gene duplication

373Whole genome duplications can result in sudden expansions in gene number

374Duplications of genes and groups of genes have occurred frequently in the past

375Box 14.2 Gene duplication and genetic redundancy

378The sequences of some duplicated genes do not diverge

380Genome evolution also involves rearrangement of existing genes

38014 .2.2 Acquisition of new genes from other species

383Box 14.3 The origins of the eukaryotic cell

38314 .3

Noncoding DNA and Genome Evolution

384Box 14 .4 The origin of a microsatellite

38414 .3 .1 Transposable elements and genome evolution

384

14 .3 .2 The origins of introns

385'Introns early' and 'introns late' : two competing hypotheses

385The current evidence disproves neither hypothesis

386Box 14.5 The role of noncoding DNA

38614.4

The Human Genome: The Last Five Million Years

387

Chapter 15 Molecular Phylogenetics

39 115.1

The Origins of Molecular Phylogenetics

392Box 15 .1 Phenetics and clap istics

39315.2

The Reconstruction of DNA-based Phylogenetic Trees

39415 .2 .1 The key features of DNA-based phylogenetic trees

394Gene trees are not the same as species trees

395

15.2 .2 Tree reconstruction

397Box 15 .2 Terminology for molecular phylogenetics

397

Sequence alignment is the essential preliminary to tree reconstruction

397

Box 15.3 Multiple substitutions

399

Converting the alignment data into a phylogenetic tree

399Assessing the accuracy of a reconstructed tree

401

Molecular clocks enable the time of divergence of ancestral sequences to b eestimated

401

15.3

The Applications of Molecular Phylogenetics

40215.3 .1 Examples of the use of phylogenetic trees

402

DNA phylogenetics has clarified the evolutionary relationships betwee nhumans and other primates

402The oldest life on Earth

402The origins of AIDS

404Problems with prions

40515 .3 .2 Molecular phylogenetics as a tool in the study of human prehistory

406Intraspecific studies require highly variable genetic loci

406Box 15.4 Haplotypes

406

The origins of modern humans - out of Africa or not?

406Box 15.5 Genes in populations

408

The patterns of more recent migrations into Europe are also controversial

410Prehistoric human migrations into the New World

410

Appendix - Keeping up to Date

41 5

Glossary

41 9

Index

453