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Contents

Abbreviations

Preface

Supplementary learning aids

Before we start - Intelligent use of the Internet

PART ONE: The Basics

Chapter 1 DNA structure and gene expression

1.11.1.1L1.2

Building blocks and chemical bonds in DNA, RNA and polypeptides

DNA, RNA and polypeptides are large polymers defined by a linear sequence of simple repeating unitsCovalent bonds confer stability; weaker noncovalent bonds facilitate intermolecular associationsand stabilize structure

DNA structure and replicationThe structure ofDNA is an antiparallel double helix

Box 1.1 Examples of the importance of hydrogen bonding in nudeic acids and proteins

DNA replication is semi-conservative and synthesis ofDNA strands is semi-discontinuous

The DNA replication machinery in mammalian cells is complex

Box 1.2 Major dasses of proteins used in the DNA replication machinery

Viral genomes are frequendy maintained by RNA replication rather than DNA replication

RNA transcription and gene expression

The f]ow of gene tic information in cells is almost exclusively one way: DNA-RNA- proteinOnly a small fraction of the DNA in complex organism~ is expressed to give a protein or RNAproduct

During transcription genetic information in some DNA segments (genes) specifies RNA

Cis-acting regulatory elements and trans-acting transcription factors are required in eukaryoticgene expression

Tissue-specific gene expression involves selective activation of specific genes

RNA processing

RNA splicing removes nonessential RNA sequences from the primary transcriptSpecialized nucleotides are added to the 5' and 3' ends of most RNA polymerase 11transcripts

Translation, post-translational processing and protein structure

During translation mRNA is decoded on ribosomes to specifY the synthesis of polypeptidesThe genetic code is degenerate and not quite a universal codePost-translational modifications include chemical modifications of some amino acids and

polypeptide cleavage

Protein secretion and intracellular export is controlled by specific localization signals or bychemical modifications

Protein structure is highly varied and not easily predicted from the amino acid sequence

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1.5.4

Chapter 2 Chromosome structure and function

1.5.5

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2.22.2.1

Ploidy and the cell cyde

Structure and function of chromosomes

Packaging of DNA into chromosomes requires multiple hierarchies of DNA folding

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Chapter 3

2.2.22.2.3

2.2.42.2.52.2.6

2.32.3.12.3.22.3.3

2.42.4.1

Individual chromosomes occupy nonoverlapping territories in an interphase nucleusChromosomes as functioning organelles: the pivotal role of the centromere

Box 2.1 The mitotic spindle and its components

Chromosomes as functioning organelles: origins of replicationChromosomes as functioning organelles: the telomeresHeterochromatin and euchromatin

Mitosis and meiosis are the two types of ceO divisionMitosis is the normal form of cell division

Meiosis is a specialized form of cell division giving rise to sperm and egg cellsX-Y pairing and the pseudoautosomal regions

Studying human chromosomesMitotic chromosomes can be seen in any dividing cell, but meiotic chromosomes are hard to study inhumans

2.4.2

Box 2.2 Chromosome banding

Molecular cytogenetics: chromosome FISH

Box 2.3 Human chromosome nomenclature

2.4.3

2.52.5.12.5.2

Chromosome painting, molecular karyotyping and comparative genome hybridization

Chromosome abnormalities

Types of chromosomal abnormalityNumerical chromosoma] abnorma]ities involve gain or ]oss of complete chromosomes

Box 2.4 Nomenclature of chromosome abnormalities

Structural chromosomal abnormalities result from misrepair of chromosome breaks or from

malfunction of the recombination system

Apparently normal chromosomal complements may be pathogenic if they have the wrongparental origin

2.5.3

2.5.4

Cells and development

3.13.1.13.1.23.1.3

The structure and diversity of ceOs

Prokaryotes and eukaryotes represent the fundamental division of cellular life formsCell size and shape can vary enormously, but rates of ditTusion fix some upper limit~In multicellular organisms, there is a fundamental distinction between somatic cells and the germ line

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Box 3.1 IntraceOular organization of animal cells 62

Box 3.2 The cytoskeleton: the key to ceO movement and cell shape and a major frameworkfor intracellular transport 64

3.1.43.1.5

3.23.2.13.2.2

In multicellular organisms, no two cells carry exactly the same DNA sequenceCells from multicellular organisms can be studied in situ or in culture

CeO interactions

Communication between cells involves the perception of signaling molecules by specific receptors

Activated receptors initiate signal transduction pathways that may involve enzyme cascades or secondmessengers, and result in the activation or inhibition of transcription factorsThe organization of cells to form tissues requires cell adhesionThe extracellular matrix provides a scatTold for all tissues in the body and is also an important

source of signals that control cell behavior

An overview of development

The specialization of ceOs during developmentCell specialization involves an irreversible series of hierarchica] decisions

Box 3.3 Animal models of development

The choice between alternative fates may depend on lineage or position

Box 3.4 Twinning in human enibryos

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3.4.3 Stem cells are se!f-renewing progenitor cells

Box 3.5 Where our tissues come from - the developmental hierarchy in mammals

Box 3.6 The diversity of human cells

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3.53.5.13.5.23.5.33.63.6.1

A variety of tissue stem cells are known to exist but much remains to be learned about them

Embryonic stem (ES) cells have the potential to form any tissueThe differentiation potential of tissue stem cells is controversial

Pattern formation in development

Emergence of the body plan is dependent on axis specification and polarizationHomeotic mutations reveal the molecular basis of positional identityPattern formation often depends on signal gradientsMorphogenesis

Morphogenesis can be driven by changes in cell shape and size

Box 3.7 Polarizing the mammalian embryo - signals and gene products

Major morphogenetic changes in the embryo result from differential cell affinityCell proliferation and programmed cell death (apoptosis) are important morphogenetic mechanisms

Early human development: fertilization to gastrulation

Fertilizatíon activates the egg and brings together the nuclei of sperm and egg to form a uniqueindividual

Cleavage partitions the zygote into many smaller cells

Only a small percentage of the cells in the early mammalian embryo gives rise to the mature organismlmplantation

Gastrulation is a dynamic process whereby cells of the epiblast give rise to the three germ layers

Box 3.8 Extra-embryonic membranes and the placenta

Box 3.9 Sex determination: genes and the environment in development

Neural development

The nervous system deve!ops after the ectoderm is induced to differentiate by the underlyingmesoderm

Pattern formation in the neural tube involves the coordinated expression of genes along two axesNeuronal differentiation involves the combinatorial activity of transcription factors

Conservation of developmental pathways

Many human diseases are caused by the failure of normal developmental processesDevelopmental processes are highly conserved at both the single gene leve! and the level ofcomplete pathways

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3.8.23.8.3

3.93.9.13.9.2

Chapter 4 Genes in pedigrees and populations

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4.24.2.14.2.2

Monogenic versus multifactorial inheritance

Mendelian pedigree patterns

Dominance and recessiveness are properties of characters, not genesThere are five basic Mendelian pedigree patterns

Box 4.1 Characteristics of the Mendelian patterns ofinheritance

The mode of inheritance can rarely be defined unambiguously in a single pedigreeOne gene-one enzyme does not imply one gene-one syndromeMitochondrial inheritance gives a recognizable matrilineal pedigree pattern

Box 4.2 The complementation test to discover whether two recessive characters aredetermined by allelic genes

Complications to the basic Mendelian pedigree patternsCommon recessive conditions can give a pseudo-dominant pedigree patternFailure of a dominant condition to manifest is called nonpenetranceMany conditions show variable expression

For imprinted genes, expression depends on parental originMale lethality may complicate X-linked pedigreesNew mutations often complica te pedigree interpretation, and can lead to mosaicism

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Genetics of multifactorial characters: the polygenic-threshold theorySome historyPolygenic theory of quantitative traits

Box 4.3 Two common misconceptions about regression to the mean

Box 4.4 Partitioning of variance

Polygenic theory of discontinuous characters

Counseling in non-Mendelian conditions uses empiric risks

Factors affecting gene frequencies

There can be a simple relation between gene frequencies and genotype frequenciesGenotype frequencies can be used (with caution) to calculate mutation rates

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Box 4.5 Hardy-Weinberg equilibrium genotype frequencies for aBele frequenciesp(Al) and q (A2) 117

Box 4.6 The Hardy- Weinberg distribution can be used (with caution) to calculate carrier

frequencies and simple risks for counseling 118

Box 4.7 Mutation-selection equilibrium 118

4.5.3 Heterozygote advamage can be much more important than recurrent mutation for determiningthe frequency of a recessive disease.

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4.5.14.5.2.

Box 4.8 Selection in favor of heterozygotes for CF

Chapter 5 Amplifying DNA: PCR and cell-based DNA cloning

5.1

5.25.2.1

The importance of DNA cloning

PCR: basic features and applicationsPrincipies of baÚc PCR and reverse transcriptase (RT) PCR

Box 5.1 A glossary ofPCR methods

PCR has two major limitations: short sizes and low yields of productsGeneral applications of PCR

Some PCR reactions are designed to permit multiple amplification products and to amplii)' previouslyuncharacterized sequences

Principies of cell-based DNA cloning

An overview of cell-based DNA doning

Box 5.2 Restriction endonucleases and modification-restriction systems

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5.3.2 Restriction endonudeases enable the target DNA to be cut into manageable pieces which canbe joined to similarly cut vector molecules 130

5.3.3 lntroducing recombinant DNA into recipient cells provides a method for fractionating a complexstarting DNA population 133

5.3.4 DNA libraries are a comprehensive set of DNA dones representing a complex starting DNApopulation 133

5.3.5 Recombinant screening is often achieved by insertional inactivation of a marker gene 135

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5.35.3.1

5.45.4.1

Box 5.3 Nonsense suppressor mutations

Box 5.4 The importance of sequence tagged sites (STSs)

Cloning systems for amplif)ring different sized fragmentsStandard plasmid vectors provide a simple way of doning small DNA fragments in bacterial (andsimple eukaryotic) cells

Lambda and cosmid vectors provide an efficient means of doning moderately large DNAfragments in bacteria] cells

Large DNA fragments can be doned in bacteria] cells using vectors based on bacteriophage Pland F factor plasmids

Yeast artificial chromosomes (YACs) enable doning of megabase fragments

Cloning systems for producing single-stranded and mutagenized DNA

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5.5.1 Single-stranded DNA for use in DNA sequencing is obtained using M13 or phagemid vectors orby linear PCR amplification

Oligonucleotide mismatch muragenesis can create a predetermined single nucleotide change inany cloned gene

PCR-based mutagenesis includes coupling of desired sequences or chemical groups to a targetsequence and site-specific mutagenesis

Cloning systems designed to express genes

Large amounes of protein can be produced by expression cloning in bacterial cel!sPhage display is a form of expression cloning in which proteins are expressed on bacterial cel! surfacesEukaryotic gene expression is carried out with greater fidelity in eukaryotic cel! lines

Box 5.5 Transferring genes into cultured animal cells

5.5.2

5.5.3

5.6

5.6.15.6.25.6.3

Chapter 6 Nucleic acid hybridization: principies and applications

6.16.1.16.1.2

Preparation of nucleic acid probes

Nucleic acids can conveniently be labeled in vitro by incorporation of modified nucleotidesNucleic acids can be labeled by isotopic and nonisotopic methods

Box 6.1 Principies of autoradiography

6.2

6.2.1Principies of nucleic acid hybridization 161

Nucleic acid hybridization is a method for identifYing closely related moleculcs within two nucleic acidpopulations 161

Box 6.2 Fluorescence labeling and detection systems 164

The kinetics of DNA reassociation are defined by the product of DNA concentration and time (Co,) 164

Box 6.3 A glossary of nucleic acid hybridization 166

A wide variety of nucleic acid hybridization assays can be used 167

Nucleic acid hybridization assays using cloned DNA probes to screen uncloned nucleicacid populations 168

Dot-blot hybridization, a rapid screening method, often employs al!ele-specific oligonucleotide probes 168

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6.2.3

6.3

6.3.1

6.3.2

Box 6.4 Standard and reverse nucleic acid hybridization assays

Southern and Northern blot hybridizations detect nucleic acids that have been size-fractionated by gelelectrophoresis

Pulsed field gel electrophoresis extends Southern hybridization to include detection of very largeDNA molecules

In in situ hybridization probes are hybridized to denatured DNA of a chromosome preparation orRNS of a tissue section fixed on a glass slide

Hybridization assays using cloned target DNA and microarrays

Colony blot and plaque lift hybridization are methods for screening separated bacterial colo niesor plaques

Gridded high density arrays of transformed cel! clones or DNA clones has greatly increased theefficiency of DNA library screening

DNA microarray technology has enormously extended the power of nucleic acid hybridization

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6.3.4

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6.4.2

6.4.3

Chapter 7 Analyzing DNA and gene structure. variation and expression

7.17.1.1

Sequencing and genotyping DNA

Standard DNA sequencing involves enzymatic DNA synehesis using base-specific dideoxynucleotidechain terminators

7.1.27.1.3

Box 7.1 Producing single-stranded DNA sequencing templates

Automated DNA sequencing and microarray-based re-sequencing

Basic genotyping of restriction site polymorphisms and variable number of tandem repeatpolymorphisms

Identifying gene s in cloned DNA and establishing their structure7.2

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PART TWO:

Box 7.2 Common dasses of DNA polymorphism which are amenable to simple genotypingmethods 187

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7.37.3.1

Exon trapping identifies expressed sequences by using an artificial RNA splicing assay

cDNA selection identifies expressed sequences in genomic dones by heteroduplex formationAchieving full-Iength cDNA sequences: overlapping done sets, and RACE-PCR amplificationMapping transcription start sites and defining exon-intron boundaries

Studying gene expressionPrincipies of expression screening

Box 7.3 Database homology searching

Hybridization-based gene expression analyses: from single gene analyses to whole genome expressionscreemng

PCR-based gene expression analyses: RT -PCR and mRNA differential displayProtein expression screens typically use highly specific antibodies

Box 7.4 Obtaining antibodies

Autofluorescent protein tags provided a powerful way of tracking subcellular localization of proteins

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7.3.37.3.4

7.3.5

Chapter 8 Genome projects and model organisms

The human genome and its relationship to other genomes

8.18.1.18.1.2

The ground-breaking importance of geno me projectsGenome projects prepared the way for systematic studies of the Universe within

The medical and scientific benefits of the genome projects are expected to be enormous

Box 8.1 A genomics glossary

Background and organization of the Human Genome ProjectDNA polymorphisms and new DNA doning technologies paved the way for sequencing our genomeThe Human Genome Project was mainly conducted in large genome centers with high-throughputsequencing capacity

How the human genome was mapped and sequencedThe first useful human genetic maps were based on microsatellite markers

Box 8.2 Human gene and DNA segment nomendature

Box 8.3 Major milestones in mapping and sequencing the human genome

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8.38.3.1

8.3.2 The first high resolution physical maps of the human genome were based on done contigs and STSlandmarks 213

Box 8.4 Hybrid cell mapping 215

8.3.3 The final stage of the Human Genome Project was crucially dependent on BAC/PAC done contigs 217

Box 8.5 Physical mapping by building done contigs 218

8.3.4 The first high density human gene maps were based on EST markers 220

8.3.5

Box 8.6 Co-operation, competition and controversy in the genome projects

The draft human genome sequence suggested 30000-35000 human genes, but getting a precisetotal is difficult

The final stages of the Human Genome Project: gene annotation and gene ontologyAnalyses of human genome sequence variation are important for anthropological andmedical research

Without proper safeguards, the Human Genome Project could lead to discrimination againstcarriers of disease genes and to a resurgence of eugenics

Genome projects for model organismsThere is a huge diversity of prokaryotic genome projects

The S.cerevisiaegenome project was the first of many successfulprotist genome projects

Box 8.7 Model unicellular organisms

The Caenorhabditiselegansgenome project was the first animal genome project to be completed

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8.4.4 Metazoan genome projects are mostly focusing on models of development and disease 229

Box 8.8 Model multicellular animals for understanding development, disease and genefunction 230

Chapter 9 Organization of the human genome

9.19.1.1

9.1.2

General organization of the human genomeAn overview of the human genome

The mitochondrial geno me consists of a small circular DNA duplex which is densely packedwith genetic information

Box 9.1 Genome copy number variation in human cells

Box 9.2 The limited autonomy of the mitochondrial genome

The nuclear genome consists of 24 different DNA molecules corresponding to the 24 different humanchromosomes

The human genome contains about 30000-35 000 unevenly distributed genes but precise numbersare uncertain

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9.1.4

9.2

9.2.1

Box 9.3 DNA methylation and CpG islands

Organization, distribution and function of human RNA genesA total of about 1200 human genes encode rRNA or tRNA and are mostly organized intolarge gene clusters

Small nuclear RNA and small nucleolar RNA are encoded by mostly dispersed, moderately large genefamilies

9.2.2

9.2.3

Box 9.4 Anticodon specificity of eukaryotic cytoplasmic tRNAs

MicroRNAs and other novel regulatory RNAs are challenging preconceptions on the extent of RNAfunction

9.39.3.19.3.2

Organization, distribution and function of human polypeptide-encoding genesHuman genes show enormous variation in size and internal organizationFunctionally similar genes are occasionally clustered in the human genome, but are more oftendispersed over different chromosomes

Box 9.5 Human genome and human gene statistics

Overlapping genes, genes-within-genes and polycistronic transcription units are occasionally foundin the human genome

Polypeptide-encoding gene families can be classified according to the degree and extent of sequencerelatedness in family members

Genes in human gene families may be organized into small clusters, widely dispersed or both

Pseudogenes, truncated gene copies and gene fragments are commonly found in multigene familiesHuman proteome classification has begun but the precise functions of many human proteins remainuncertain

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Tandemly repeated noncoding DNA

Satellite DNA consists of very long arrays of tandem repeats and can be separated from bulk DNAby density gradient centrifugationMinisatellite DNA is composed of moderately sized arrays of tandem repeats and is often locatedat or close to telomeres

Microsatellite DNA consists of short arrays of simple tandem repeats and is dispersed throughoutthe human genome

Interspersed repetitive noncoding DNA

Transposon-derived repeats make up > 40% of the human genome and mostly arose through RNAintermediates

Some human UNE 1 elements are actively transposing and enable transposition of SINES, processedpseudogenes and retrogenes

Alu repeats occur more than once every 3 kb in the human genome and may be subject topositive selection

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Chapter 10 Human gene expression 275

10.1 An overview of gene expression in human ceUs 276

Box 10.1 Spatial and temporal restriction of gene expression in mammalian cells 276

10.2 Control of gene expression by binding of trans-acting protein factors to cis-acting regulatorysequences in DNA and RNA 277

10.2.1 Hisrone modificarion and chromatin remodeling facilirare access ro chromarin by DNA-binding facrors 27810.2.2 Ubiquirous rranscriprion facrors are required for rranscriprion by RNA polymcrases l and 1Il 279

10.2.3 Transcriprion by RNA polymerase II requires complex sers of cis-acring regularory sequences andrissue-specific rranscriprion facrors 280

10.2.4. Transcriprion facrors conrain conserved srrucrural motifs rhar permir DNA binding 282

Box 10.2 Classes of cis-acting sequence elements involved in regulating transcription ofpolypeptide-encoding genes 283

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10.2.6

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A variery of mechanisms permir rranscriptional regulation of gene expression in response roexrernal srimuli

Translarional control of gene expression can involve recognirion of UTR regularory sequences byRNA-binding proreins

AIternative transcription and processing of individual genesThe use of alternarive promorers can generare rissue-specific isoforms

Human genes are prone ro alrernarive splicing and alrernative polyadenylationRNA ediring is a rare form of processing whereby base-specific changes are inrroduced into RNA

Box 10.3 AIternative splicing can alter the functional properties of a protein

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Differential gene expression: origins through asymmetry and perpetuation throughepigenetic mechanisms such as DNA methylation 294

Selecrive gene expression in cells of mammalian embryos mosr likely develops in response ro shorrrange cell-cell signaling evenrs 295

DNA merhylarion is an imporrant epigeneric facror in perperuaring gene repression in verrebrare cells 295Animal DNA merhylation may provide defense against transposons as well as regulating geneexpression 297

Long range control of gene expression and imprinting 298Chromarin structure may exert long-range control over gene expression 298

Expression of individual genes in gene clusters may be co-ordinared by a common locus conrrol region 299Some human genes show selective expression of only one of the rwo parental alleles 300Genomic imprinting involves ditTerences in rhe expression of alleles according to parent of origin 301

Box lOA Mechanisms resulting in monoallelic expression from biaUelic genes in human cells 302

Box 10.5 The nonequivalence of the maternal and paternal genomes 302

The mechanism of genomic imprinring is unclear but a key component appears ro be DNAmethylation 303

X chromosome inactivation in mammals involves very long range cis-acting repression of geneexpression 305

The unique organization and expression of Ig and TCR genes 306DNA rearrangements in B and T cells generate cell-specific exons encoding Ig and TCR variablere~ns ~8

Heavy chain class switching involves joining of a single VD) exon ro alternative constant regiontranscription unirs 309

The monospecificiry of Igs and TCRs is due to allelic and light chain exclusion 310

Chapter 11 Instability of the human genome: mutation and DNA repair 315

11.1 An overview of mutation, polymorphism, and DNA repair

11.2 Simple mutations

11.2.1 Mutations due to errors in DNA replication and repair are frequenr

Box 11.1 Classes of genetic polymorphisms and sequence variation

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The frequency of individual base substitutions is nonrandom according to substitution class

The frequency and spectrum of mutations in coding DNA differs from that in noncoding DNA

Box 11.2 Mechanisms that affect the population frequency of aUeles

The location ofbase substitutions in coding DNA is nonrandom

Box 11.3 Classes of single base substitution in polypeptide-encoding DNA

11.2.5 Substitution rates vary considerably between different genes and between different gene components11.2.6 The substitution rate can vary in differcnt chromosomal regions and in different lineages

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Box 11.4 Sex differences in mutation rate and the question of male-driven evolution

Genetic mechanisms which result in sequence exchanges between repeatsReplication slippage can cause VNTR polymorphism at short tandem repeats (microsatellites)

Large units of tandemly repcated DNA are prone to insertion/ deletion as a result of unequalcrossover or unequal sister chromatid exchangesGene conversion events may be rclatively freql1cnt in tandemly repetitive DNA

Pathogenic mutationsThere is a high deleterious mutation rate in hominids

The mitochondrial genome is a hotspot for pathogenic mutationsMost splicing mutations alter a conserved sequence nceded for normal splicing, but some occurin sequences not normally required for splicingMutations that introduce a premature termination codon often result in unstable mRNA butother outcomes are possible

The pathogenic potential of repeated sequences

Slipped strand mispairing of short tandem repeats predisposes to pathogenic deletions andframeshifting insertions

Unstable expansion of short tandem repeats can cause a variery of diseases but the mutationalmechanism is not well understood

Tandemly repeated and clustered gene families may be prone to pathogenic unequal crossover andgene conversion-like events

lnterspersed repeats often predispose to large deletions and duplications

Pathogenic inversions can be produced by intrachromatid recombination between inverted repeatsDNA seql1ence transposition is not uncommon and can cause disease

DNA repair

DNA repair usually involves cutting out and resynthesizing a whole area ofDNA surrounding thedamage

DNA repair systems share components and processes with the transcription and recombinationmachinery

Hypersensitivity to agents that damage DNA is often the result of an impaired cellular responseto DNA damage, rather than defective DNA repair

Chapter 12 Our place in the tree of life

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Evolution ofgene structure and duplicated genes

Spliceosomal introns probably originated from group 11introns and first appeared in early eukaryoticcells

Complex genes can evolve by intragenic duplication, often as a result of exon duplication

Box 12.1 Intron groups

Exon shuffiing can bring together new combinations of protein domains

Gene duplication has played a crucially important role in the evolution of multicellular organismsThe globin superfamily has evolved by a process of gene duplications, gene conversions, and genelosslinactivation

Box 12.2 Symmetrical exons and intron phases

Box 12.3 Gene duplication mechanisms and paralogy

12.1.6 Retrotransposition can permit exon shuffiing and is an important contributor to gene evolution

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Evolution of chromosomes and genomesThe mitochondrial genome may have originated following endocytosis of a prokaryotic cell by a

eukaryotic cell precursor

Box 12.4 The universal tree of life and horizontal gene transfer

Reduced selection pressure caused the mitochondrial genetic code to divergeThe evolution of vertebrate genomes may have involved whole genome duplicationThere have be en numerous major chromosome rearrangements during the evolution of mammalian

genomesSegmental duplication in primate lineages and the evolutionary instability of pericentromeric andsubtelomeric sequencesThe human X and Y chromosomes exhibit substantial regions of sequence homology, including

common pseudoautosomal regionsHuman sex chromosomes evolved from autosomes and diverged due to periodic regional

suppression of recombinationSex chromosome differentiation results in progressive Y chromosome degeneration and Xchromosome inactivation

Molecular phylogenetics and comparative genomicsMolecular phylogenetics uses sequence alignments to construct evolutionary trees

New computer programs align large scale and whole genome sequences, aiding evolutionary analysesand identification of conserved sequencesGene number is generally proportional to biological complexityThe extent of progressive protein specialization is being revealed by proteome comparisons

What makes us human?What makes us different from mice?

What makes us different from our nearcst relatives, the great apes?

Box 12.5 A glossary of common metazoan phylogenetic groups and terms

12.5 Evolution of human populations12.5.1 Genetic evidence has suggested a recent origin of modern humans from African populations12.5.2 Human genetic diversity is low and is mostly due to variation within populations rather than

between them

Box 12.6 Coalescence analyses

PART THREE: Mapping and identifying disease genes and mutations

Chapter 13 Genetic mapping of Mendelian characters

13.113.1.113.1.213.1.313.1.413.1.5

13.213.2.113.2.2

Recombinants and nonrecombinants

The recombination fraction is a measure of genetic distanceRecombination fractions do not exceed 0.5 however great the physical distance

Mapping functions define the relationship between recombination fraction and genetic distanceChiasma counts and total map lengthPhysical vs. genetic maps: the distribution of recombinants

Genetic markers

Mapping human disease genes requires genetic markersThe heterozygosity or polymorphism information content measure how informative a marker is

Box 13.1 The development of human genetic markers

13.2.3. DNA polymorphisms are the basis of all current genetic markers

Box 13.2 Informative and uninformative meioses

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13.3.2

Two-point mappingScoring recombinants in human pedigrees is not always simple

Computerized lod score analysis is the best way to analyze complex pedigrees for linkagebetween Mendelian characters

Box 13.3 Calculation of lod scores for the families in Figure 13.6

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CONTENTS I xv

Lod scores of +3 and -2 are the criteria for linkage and exdusion (for a single test)For whole genome searches a genome-wide threshold of significance must be used

Multipoint mapping is more efficient than two-point mappingMultipoint linkage can loca te a disease locus on a framework of markersMarker framework maps: the CEPH families

Box 13.4 Bayesian calculation of linkage threshold

Multipoint disease-marker mapping

Fine-mapping using extended pedigrees and ancestral haplotypesAutozygosity mapping can map recessive conditions efficiently in extended inbred families

Identifying shared ancestral segments allowed high-resolution mapping of the loci for cysticfibrosis and Nijmegen breakage syndrome

Standard lod score analysis is not without problemsErrors in genotyping and misdiagnoses can generate spurious recombinantsComputational difficulties limit the pedigrees that can be analyzedLocus heterogeneity is always a pitfall in human gene mappingMeiotic mapping has limited resolution

Characters whose inheritance is not Mendelian are not amenable to mapping by the methodsdescribed in this chapter

Chapter 14 IdentifYing human disease genes

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14.214.2.114.2.214.2.3

14.314.3.114.3.214.3.314.3.4

14.3.5

14.414.4.1

PrincipIes and strategies in identifYing disease genes

Position-independent strategies for identifYing disease genes

Identifying a disease gene through knowing the protein productIdentifying the disease gene through an animal model

Identification of a disease gene using position-independent DNA sequence knowledge

Positional doning

The first step is to define the candidate region as tightly as possibleA contig of dones must be established across the candidate regionA transcript map defines all genes within the candidate regionGenes from the candidate region must be prioritized for mutation testing

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Box 14.1 Transcript mapping: laboratory methods that supplement database analysis foridentifYing expressed sequences within genomic dones 421

The special relevance of mouse mutants

Use of chromosomal abnormalities

Patients with a balanced chromosomal abnormality and an unexplained phenotype are interesting

Box 14.2 Mapping mouse genes

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14.5.1

14.5.2

14.6

14.6.114.6.214.6.314.6.414.6.5

Box 14.3 Pointers to the presence of chromosome abnormalities

Box 14.4 Position effects - a pitfall in disease gene identification

Confirming a candidate geneMutation screening to confirm a candidate gene

Box 14.5 CGH for detecting submicroscopic chromosomal imbalances

Once a candidate gene is confirmed, the next step is to understand its function

Eight examples iIIustrate various ways disease genes have been identified

Direct identification of a gene through a chromosome abnormality: Sotos syndromePure transcript mapping:Treacher Collins syndrome

Large-scale sequencing and search for homologs: branchio-oto-renal syndromePositional candidates defined by function: rhodopsin and fibrillin

A positional candidate identified through comparison of the human and mouse maps: PAX3and Waardenburg syndrome

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14.6.614.6.714.6.S

Inference from function i/1 vilr,,: Fanconi anemia

Inference from function i/1vivo: myosin 15 and DFNB3 deafness

Inference from the exprcssion pattern: otoferlin

15.1

Chapter 15 Mapping and identifying genes conferring susceptibility to complex diseases

Deciding whether a non-Mendelian character is genetic: the role of family, twin andadoption studiesThe A value is a measure of familial clusteringThe importance of shared family environmentTwin studies sutTer from many limitations

Adoption studies: the gold standard for disentangling genetic and environmental factors

Segregation analysis allows analysis of characters that are anywhere on the spectrumbetween purely Mendelian and purely polygenicBias of ascertainment is often a problem with family data: the example of autosomal recessiveconditions

Complex segregation analysis is a general method for estimating the most likely mix of geneticfactors in pooled family data

15.3 Linkage analysis of complex characters15.3.1 Standard lod score analysis is usually inappropriate for non-Mcndelian characters

Box 15.1 Correcting the segregation ratio

Non-paramctric linkage analysis does not require a genetic model

Shared segment analysis in families: atTected sib pair and atTected pcdigree member analysisThresholds of significance are an important consideration in analysis of complex diseases

Association studies and linkage disequilibriumWhy associations happen

Association is in principIe quite distinct from linkage, but whcre the family and the populationmerge, linkage and association merge

Box 15.2 Measures of linkage disequilibrium

Many studies show islands of linkage disequilibrium separated by recombination hotspotsDcsign of association studiesBox 15.3 The transmission disequilibrium test (TDT) to determine whether marker allele

M1 is associated with a disease

15.4.5 Linkage and association: complcmentary techniques

15.1.115.1.215.1.315.1.4

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15.5 Identifying the susceptibility alleles

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Box 15.4 Sample sizes needed to find a disease susceptibility locus by a whole genomescan using either atTected sib pairs (ASP) or the transmission disequilibrium test (TDT) 447

Eight examples iIIustrate the varying success of genetic dissection of complex di seas esBreast cancer: identifying a Mendelian subset has led to important medical advances, but do es notcxplain the causes of the common sporadic diseaseHirschsprung disease: an oligogenic diseaseAlzheimer diseasc: genetic factors are important both in the common late-onset form and in the rare

Mendelian early-onset forms, but they are ditTerent genes, acting in ditTerent waysType 1 diabetes mellitus: still the geneticist's nightmare?

Ethics Box 1 Alzheimer disease, ApoE testing and discrimination

Type 2 diabetes: two susceptibility factors, one so common as to be undetectable by linkage; theother very complex and in certain populations onlyIntlammatory bowcl disease: a clear-cut susceptibility gene identifiedSchizophrenia: the special problems of psychiatric or behavioral disordersObesity: genetíc analysis of a quantitative trait

Overview' andsummaryWhy is it so difficult?

If it all works out and we idcntify susceptibility alleles-then what?

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CONTENTS I xvii

Chapter 16 Molecular pathology

16.1

16.216.316.3.1

16.3.316.3.4

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16.4.216.4.3

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16.516.5.116.5.216.5.3

Introduction

The convenient nomenclature of d and 1! alleles hides a vast diversity of DNA sequencesA first classification of mutations is into 10ss of function vs. gain of function mutationsFor molecular pathology, the important thing is not the sequence of a mutant allele but its etTect

Box 16.1 The main classes of mutation

Box 16.2 Nomenclature for describing sequence changes

16.3.2 Loss of function is likely when point mutations in a gene produce the same pathological changeas dcletions

Box 16.3 A nomenclature for describing the effect of an allele

Gain of function is likely when only a specific mutation in a gene produces a given pathologyDeciding whether a DNA sequence change is pathogenic can be difficult

Loss of function mutations

Many ditTerent changes to a gene can cause loss of function

Box 16.4 Hemoglobinopathies

Box 16.5 Guidelines for assessing the significance of a DNA sequence change

In haploinsufficiency a 50% reduction in the level of gene function causes an abnormal phenotypeMutations in proteins that work as dimers or multimers sometimes produce dominant negativeetTects

Epigenetic modification can abolish gene function even without a DNA sequence change

Gain of function mutations

Acquisition of a novel function is rare in inherited disease but common in cancer

Overexpression may be pathogenicQualitative changes in a gene product can cause gain of function

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16.6 Molecular pathology: from gene to disease 471

16.6.1 For loss of function mutations the phenotypic etTect depends on the residuallevcl of gene function 471

Box 16.6 Molecular pathology ofPrader-Willi and Angelman syndromes 472

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16.716.7.116.7.216.7.3

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16.816.8.116.8.2

Loss of function and gain of function mutations in the same gene will cause ditTerent diseasesVariability within families is evidence of modifier genes or chance etTectsUnstable expanding repeats - a novel cause of disease

Protein aggregation is a common pathogenic mechanism in gain of function diseases

For mitochondrial mutations, heteroplasmy and instability complicate the relationship betweengenotype and phenotype

Molecular pathology: from disease to geneThe gene underlying a disease may not be the obvious oneLocus heterogeneity is the rule rather than the exception

Mutations in ditTerent l11embers of a gene family can produce a series of related or overlappingsyndromes

Clinical and molecular classifications are alternative tools for thinking about diseases, and each isvalid in its own sphere

Molecular pathology of chromosomal disorders

Microdcletion syndromes bridge the gap between single gene and chrol11osol11alsyndrol11es

The major etTects of chromosomal aneuploidies may be caused by dosage imbalances in a fewidentifiable genes

Chapter 17 Cancer genetics

17.1

17.2

17.317.3.1

Introduction

The evolution of cancer-;~-J~' UNr~ER§mÁDCES

,¡¡;j'1l11 ,,-;:-_. -. 1 ..,.~ vil \~omprv;iÚS(J "0;. la 1::..w.:(/encwOncogenes

The history of oncogenesBIBLIOTECAFUNDADORES

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Box 17.1 Two ways of making a series of successive mutations more likely

17.3.2 The functions of oncogenes17.3.3 Activation of proto-oncogenes

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17.8

Tumor suppressor genes

The retinoblastoma paradigm

Loss of heterozygosity (LoH) screening is widely used for trying to identify TS gene locationsTumor suppressor genes are often silenced epigenetically by methylation

Stability of the genomeChromosomal instabilityDNA repair defects and DNA-level instabilityHereditary nonpolyposis colon cancer and microsatellite instabilityp53 and apoptosis

Control of the cell cycleThe Gl-S checkpoint

Integrating the data: pathways and capabilitiesPathways in colorectal cancer

A successful tumor must acquire six specific capabilities

What use is all this knowledge?

Chapter 18 Genetic testing in individuals and populations

18.1

18.2

18.318.3.118.3.218.3.318.3.418.3.518.3.6

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Introduction

The choice of material to test: DNA, RNA or protein

Scanning a gene for mutations

Methods based on sequencing

Methods based on detecting mismatches or heteroduplexesMethods based on single-strand conformation analysisMethods based on translation: the protein truncation testMethods for detecting deletionsMethods for detecting DNA methylation patterns

Testing for a specified sequence changeMany simple methods are available for genotyping a specified variant

Box 18.1 Multiplex amplifiable probe hybridization (MAPH)

Methods for high-throughput genotypingGenetic testing for triplet repeat diseases

Geographical origin is an important consideration for some tests

Gene trackingGene tracking involves three logical steps

Box 8.2 1\vo methods for high-throughput genotyping

Recombination sets a fundamentallimit on the accuracy of gene trackingCalculating risks in gene tracking

Box 18.3 The logic of gene tracking

The special problems of Duchenne muscular dystrophy

Population screeningAcceptable screening programs must fit certain criteria

Box 18.4 Use of Bayes' theorem for combining probabilities

Specificity and sensitivity measure the technical performance of a screening testOrganization of a genetic screening program

DNA profiling can be used for identifYing individuals and determining relationships

A variety of ditferent DNA polymorphisms have been used for profilingDNA profiling can be used to determine the zygosity of twinsDNA profiling can be used to disprove or establish paternity

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18.7.4 DNA profiling is a powerful tool for forensic investigations 535

Box 18.5 The Prosecutor's FaUacy 535

PART FOUR: New horizons: into the 21st century

Chapter 19 Beyond the genome project: functional genomics, proteomics and bioinformatics

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An overview of functional genomicsThe information obtained from the structural phase of the Human Genome Project is of limiteduse without functional annotation

The functions of individual genes can be described at the biochemical, cellular and whole-organismlevels

Functional relationships among genes must be studied at the levels of the transcriptome andproteome

Box 19.1 The function of glucokinase

High-throughput analysis techniques and bioinformatics are the enabling technologies offunctional genomics

Functional annotation by sequence comparison

Tentative gene functions can be assigned by sequence comparisonConsensus search methods can extend the number of homologous relationships identifiedSimilarities and ditTerences between genomes indica te conserved and functionally important sequencesComparative genomics can be exploited to identifY and characterize human disease genesA stubborn minority of genes resist functional annotation by homology searching

Global mRNA profiling (transcriptomics)Transcriptome analysis reveals how changes in patterns of gene expression coordinate the biochemicalactivities of the cell in health and disease

Direct sequence sampling is a statistical method for determining the relative abundances ofditTerent transcripts

Box 19.2 Sequence sampling techniques for the global analysis of gene expression

DNA microarrays use multiplex hybridization assays to measure the abundances of thousands oftranscripts simultaneously

The analysis of DNA array data involves the creation of a distance matrix and the clustering of relateddatapoints using reiterative algorithms

DNA arrays have been used to study global gene expression in human celllines, tissue biopsies andanimal disease models

Proteomics

Proteomics encompasses the analysis of protein expression, protein structure and protein interactionsExpression proteomics has tlourished through the combination of two major technology platforms: two-dimensional gel electrophoresis (2DGE) and mass spectrometry

Box 19.3 Protein chips

Box 19.4 Mass spectrometry in proteomics

Expression proteomics has been used to study changes in the proteome associated with diseaseand toxicity

Protein structures provide important functional informationThere are many ditTerent ways to study individual protein interactions

Box 19.5 Determination of protein structures

High throughput interaction screening using library-based methods

Box 19.6 Structural classification of proteins

The challenge of interaction proteomics is to assemble a functional interaction map of the cell

lnformation abol1l protein interactions with smallligands can improve our understanding ofbiomolecular processes and provides a rational basis for the design of drugs

Summary

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Chapter 20 Genetic manipulation of cells and animals

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20.2.520.2.620.2.7

An overview of gene transfer technology

Principies of gene transfer

Gene transfer can be used to introduce new, functional DNA sequences into cultured animalcells either transiently or stably

The production of transgenic animals requires stable gene transfer to the germ line

Box 20.1 Methods of gene transfer to animal cells in culture

Box 20.2 Selectable markers for animal cells

Box 20.3 Isolation and manipulation of mammalian embryonic stem cells

The control of transgene expression is an important consideration in any gene transfer experimentGene transfer can also be used to produce defined mutations and disrupt the expression ofendogenous genes

Gene targeting allows the production of animals carrying defined mutations in every cellSite-specific recombination allows conditional gene inactivation and chromosome engineeringTransgenic strategies can be used to inhibit endogenous gene function

20.3 Using gene transfer to study gene expression and function

20.3.1 Gene expression and regulation can be investigated using reporter genes

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20.3.2 Gene function can be investigated by generating loss-of-function and gain-of-function mutations andphenocopies 595

20.3.3 The large scale analysis of gene function by insertional mutagenesis and systematic RNA interferenceare cornerstones of functional genomics 597

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20.4.4

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Box 20.5 Sophisticated vectors used for insertional mutagenesis 599

Creating disease models using gene transfer and gene targeting technology 599Modeling disease pathogenesis and drug treatment in cell culture 599

It may be difficult to identify animal disease models generated spontaneously or induced by randommutagenesis 600

Mice have been widely used as animal models of human disease largely because specific mutations can becreated at a predetermined locus 602

Loss-of-function mutations can be modeled by gene targeting, and gain-of-function mutations by theexpression of dominant mutant genes 602

Box 20.6 The potential of animals for modeling human disease 603

1ncreasing attention is being focused on the use of transgenic animals to model complex disorders 604Mouse models of human disease may be difficult to construct because of a variety of human/ mousedifferences 605

Chapter 21 New approaches to treating disease

21.1

21.2

21.3

21.3.121.3.221.3.321.3.4

21.4

21.521.5.1

Treatment of genetic disease is not the same as genetic treatment of disease

Treatment of genetic disease

Using genetic knowledge to improve existing treatments and develop new versions ofconventional treatments

Pharmacogenetics promises to increase the effectiveness of drugs and reduce dangerous side effectsDrug companies have invested heavily in genomics to try to identify new drug targetsCell-based treatments promise to transform the potential of transplantationRecombinant proteins and vaccines

Ethics Box 1 The ethics of human cloning

Principies of gene therapy

Methods for inserting and expressing a gene in a target cell or tissue

Genes can be transferred to the recipient cells in the laboratory (ex vivo) or within the patient'sbody (in vivo)

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21.5.2 Constructs may be designed to integrate into the host ceIl chromosomes or to remain as episomes 616

Ethics Box 2 Germ line versus soma tic gene therapy 617

Box 21.1 1995 NIH Panel report on gene therapy (Orkin-Motulsky report) 619

Ethics Box 3 Designer babies 619

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21.621.6.121.6.221.6.321.6.4

21.721.7.121.7.221.7.321.7.421.7.5

Glossary

Disease index

Index

Viruses are the most commonly used vectors for gene therapy

Nonviral vector systems avoid many of the safety problems of recombinant viruses, but gene transferrates are generaIly low

Methods for repairing or inactivating a pathogenic gene in a ceIl or tissueRepairing a mutant aIlele by homologous recombinationInhibition of translation by antisense oligonucleotidesSelective destruction or repair of mRNA by a ribozymeSelective inhibition of the mutant aIlcle by RNA interference (RNAi)

Some examples of attempts at human gene therapy

The first definite success: a cure for X-linked severe combined immunodeficiencyAttempts at gene therapy for cystic fibrosisAttempts at gene therapy for Duchenne muscular dystrophyGene therapy for cancerGene therapy for infectious disease: HIV

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