GENOMES 2 EDITION
`
`SECOND
`
`T.A.BROWN
`Department of Biomolecular Sciences, UMIST, Manchester, M60 I QD, UK
`
`@WILEY-LISS
`
`A JOHN WILEY & SONS, INC. , PUBLICATION
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`00001
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`EX1072
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`

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`Published by John Wiley & Sons, Inc., by arrangement with BIOS Scientific
`Publishers Limited, 9 Newtec Place, Magdalen Road, Oxford OX4 I RE, UK
`
`This edition published in the United States of America, its dependent territories, Central and
`South America, Canada.Australia, Brunei, Cambodia, Hong Kong, India, Indonesia, Laos, Macau,
`Malaysia, New Zealand, People's Republic of China, Philippines, Singapore, South Korea, Taiwan,
`Thailand, the South Pacific and Vietnam only and not for export therefrom.
`
`© Bios Scientific Publishers Limited 2002
`
`First published 1999
`Second Edition 2002
`
`All rights reserved. No part of this book may be reproduced or transmitted, in any form or by
`any means, without permission.
`
`A CIP catalogue record for this book is available from the British Library.
`
`ISBN 0-471-25046-5
`
`Ubrary of congress Cataloging-in-Publication Data
`
`Genomes I edited by Terence A. Brown. -- 2nd ed.
`p. ;cm.
`Rev. ed. of: Genomes / T.A. Brown. 1999.
`Includes bibliographical references and index
`ISBN 0-471-25046-5 (alk. paper)
`I. Genomes.
`[DNLM: I. Genome. QH 477 G33605 2002)
`QH447 .B76 2002
`572.8' 6--dc2 I
`
`I. Brown., T.A. (Terence A.)
`
`2002003471
`
`USA
`John Wiley & Sons Inc.,
`605 Third Avenue, New York,
`NY 10158-0012, USA
`
`Canada
`John Wiley & Sons (Canada) Ltd,
`22 Worcester Road, Rexdale,
`Ontario M9W IL I, Canada
`
`Project Manager: Helen Barham PhD
`Production Editor: Sarah Carlson
`Designed, typeset and illustrated by J&L Composition Ltd, Filey, North Yorkshire, UK
`Printed by Ajanta Offset, New Delhi, India
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`00002
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`Contents
`
`Abbreviations
`
`Preface to the Second Edition
`Preface to the First Edition
`An Introduction to Genomes
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`PART I
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`Genomes, Transcriptomes and Proteomes
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`Chapter I The Human Genome
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`DNA
`I.I
`I. LI Genes are made of DNA
`Bacterial genes are made of DNA
`Virus genes are made of DNA
`1. 1.2 The structure of DNA
`Nucleotides and polynucleotides
`RNA
`I. 1.3 The double helix
`The evidence that led to the double helix
`The key features of the double helix
`Box I.I : Base-pair ing in RNA
`The double helix has structural fiexibility
`Box 1.2: Un its o f length fo r DNA molecules
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`The Human Genome
`1.2
`1.2. 1 The content of the human nuclear genome
`Genes and related sequences
`The functions of human genes
`Box 1.3: How many genes are t here in the hu ma n ge nome?
`Pseudogenes and other evolutionary relics
`Genome-wide repeats and microsatellrtes
`Box I .4: The organization o f the human ge no me
`1.2.2 The human mitochondrial genome
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`1.3 Why is the Human Genome Project Important?
`
`Study Aids
`
`Chapter 2 Genome Anatomies
`
`An Overview of Genome Anatomies
`2.1
`2. 1.1 Genomes of eukaryotes
`2.1 .2 Genomes of prokaryotes
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`2.2
`2.2.1
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`The Anatomy of the Eukaryotic Genome
`Euka ryotic nuclear genomes
`Packaging of DNA into chromosomes
`Technical Note 2. 1: Agarose gel electro pho resis
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`viii CO NTENTS
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`The special features of metaphase chromosomes
`Box 2. 1: Unusual chrom osome types
`Where are the genes in a e uka,-yotic genome?
`What genes are present in a eukaryotic genome ?
`Technical N ote 2.2: Ul t r:1ce ntrifuga tion techniques
`Families of genes
`Box 2.2: Two examples of um,su ~I ge ne o rganization
`Eukaryotic organelle genomes
`Physical featu res of orga nelle genomes
`The genetic content of organelle genomes
`The origins of organelle genomes
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`2.2.2
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`2.3
`2.3.1
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`The Anatomy of the Prokaryotic Genome
`The physical structure of the prokaryotic genome
`The traditional view of the bacterial 'chromosome'
`Complications on the E. coli theme
`Research Briefing 2. 1: Supercoiled domains in the Escherichia coli nucleoid
`2.3.2 The genetic organization of the prokaryotic genome
`Operons are characteristic features of prokaryotic genomes
`Prokaryotic genomes and the species concept
`Box 2.3: Mechanisms fo r gene flow becween prokaryo tes
`Speculation on the minimal genome content-and the identity of distinctiveness genes
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`2.4
`2.4.1
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`2.4.2
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`The Repetitive DNA Content of Genomes
`Tandemly repeated DNA
`Satellite DNA is found at centromeres and elsewhere in eukaryotic chromosomes
`Minisatellites and microsatellites
`Interspersed genome-wide repeats
`Transposition via an RNA inte rmediate
`DNA transposons
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`Study Aids
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`Chapter 3 Transcriptomes and Proteomes
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`3.1
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`Genome Expression in Outline
`Box 3. 1: Cross-references to Part 3 of Genomes
`The RNA Content of the Cell
`3.2
`3.2.1 Coding and non-coding RNA
`Box 3.2: Non-coding RNA specified by the human genome
`3.2.2 Synth~sis of RNA
`Processing of precursor RNA
`3.2.3 The transcriptome
`Studies of the yeast transcriptome
`The human transcriptome
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`The Protein Content of the Cell
`3.3
`3.3. 1 Protein structure
`The four levels of protein structure
`Amino acid diversity underlies protein diversity
`Box 3.3: No n-covalent bonds in proteins
`3.3.2 The link becween ·the transcriptome and the proteome
`The genetic code specifies how an mRNA sequence is translated into a polypeptide
`Research Briefing 3. 1: Elucidation of the genetic code
`The genetic code is not universal
`Box 3.4: The origin and evolutio n of the genetic code
`3.3.3 The link becween the proteome and the biochemistr y of the cell
`The amino acid sequence of a protein determines its function
`The multiplicity of prote_in function
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`Study Aids
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`PART 2
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`Studying Genomes
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`Chapte r 4
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`Studying DNA
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`4. 1
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`Enzym es for DNA Manipulation
`Technical Note 4.1: DNA labeling
`4.1.1 DNA polymerases
`The mode of action of a template-dependent DNA polymerase
`The types of DNA polymerases used in research
`4.1.2 Nucleases
`Restriction endonucleases enable DNA molecules to be cut at defined positions
`Examining the results of a restriction digest
`4.1.3 DNA ligases
`4.1.4 End-modification enzymes
`DNA Cloning
`4 .2
`4.2. I Cloning vectors and the way they are used
`Vectors based on E. coli plasmids
`Techn ici l Noce 4.2: DNA purification
`Cloning vectors based on E coli bacteriophage genomes
`Vectors for longer pieces of DNA
`Tec hni cal Note 4.3: Wo r ki ng with a clone library
`Cloning in organisms other than E. coli
`The Polymerase Chain Reaction (PCR)
`Technical Note 4.4: Techniques for studying RNA
`4.3.1 Carrying out a PCR
`4.3.2 The applications of PCR
`Study Aids
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`4.3
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`Chapter 5 Mapping Genomes
`
`5.2.3
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`5.2.4
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`Genetic and Physical Maps
`5.1
`5.2 Genetic Mapping
`5.2.1 Genes were the first markers to be used
`5.2.2 DNA markers for genetic mapping
`Restriction fragment length polymorphisms (RFLPs)
`Simple sequence length polymorphisms (SSLPs)
`Single nucleotide polymorphisms (SNPs)
`Box 5. 1: Why do SNPs have only two alleles 1
`Technical Note 5.1: DNA microarrays and chips
`Linkage analysis is the basis of genetic mapping
`The principles of inheritance and the discovery of linkage
`Partial linkage is explained by the behavior of chromosomes during meiosis
`From partial linkage to genetic mapping
`Linkage analysis with different types of organism
`Linkage analysis when planned breeding experiments are possible
`Box 5.2: Multipoint crosses
`Gene mapping by human pedigree analysis
`Genetic mapping in bacteria
`Physical Mapping
`5.3
`5.3. 1 Restriction mapping
`The basic methodology for restriction mapping
`The scale of restriction mapping is limited by the sizes of the restriction fragments
`Direct examination of DNA molecules for restriction sites
`5.3.2 Fluorescent in situ hybridization (FISH)
`In situ hybridization with radioactive or fluorescent probes
`FISH in action
`5.3.3 Sequence tagged site (STS) mapping
`Any unique DNA sequence can be used as an STS
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`CONTENTS
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`Fragments of DNA for STS mapping
`Re~e3;ch 81 iefing 5.1: The radiation hybrid map of the mouse genome
`A clone library can also be used as the mapping reagent for STS analysis
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`St,idyAids
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`Cha pt er 6 Sequencing Genomes
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`6. 1
`6.1.1
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`6. 1.2
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`6.2
`6.2.1
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`6.2.2
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`6.2.3
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`6.3
`6.3.1
`6.3.2
`6.3.3
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`T he Methodology for D NA Sequencing
`Chain termination DNA sequencing
`Chain temrination sequencing in outline
`Technical Note 6. 1: Polyacrylamide ge l electrophoresis
`Sox 6.1 : DNA polymecascs for chain termination sequen cing
`Chain temrination sequencing requires a single-stranded DNA template
`The primer detemrines the region of the template DNA that will be sequenced
`Themral cycle sequencing offers an alternative to t he t raditional methodology
`Fluorescent primers are the basis of automated sequence reading
`Box 6.2: The chemical degradation seq uencing method
`Departures from conventional DNA sequencing
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`Assembly of a Contiguous DNA Sequence
`Sequence assembly by the shotgun approach
`The potential of t he shotgun approach was proven by the Haemophilus influenzae sequence
`Sequence assembly by the clone contig approach
`Clone contigs can be built up by chromosome walking, but the method is laborious
`Newer more rapid methods for clone contig assembly
`Whole-genome shotgun sequencing
`Key features of whole-genome shotgun sequencing
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`The Human Genome Projects
`The mapping phase of the Human Genome Project
`Sequencing the human genome
`The future of the human genome projects
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`Study Aids
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`Chapter 7 Understanding a Genome Sequence
`
`7.1
`Locating the Genes in a Genome Sequence
`7. 1.1 Gene location by sequence inspection
`The coding regions of genes are open reading fram es
`Simple ORF scans are less effective with higher eukaryotic DNA
`Homology searches give an extra dimension to sequence inspection
`7.1.2 Experimental techniques for gene location
`Hybridization tests can determine if a fragment contains transcribed sequences
`cDNA sequencing enables genes to be mapped wit hin DNA fragments
`Methods are available for precise mapping of the ends of t ranscripts
`Exon-intron boundaries can also be located with precision
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`Determining the Functions of Individual Genes
`7.2
`7.2.1 Computer analysis of gene function
`Homology reflects evolutionary relationships
`Homology analysis can provide infomration on the function of an entire gene or of ~egments within it
`Homology analysis in the yeast genome project
`7.2.2 Assigning gene function by experimental analysis
`Functional analysis by gene inactivation
`Individual genes can be inactivated by homologous recombination
`Gene inactivation without homologous recombination
`Box 7. 1: The phenotypic effect of gene inactivation is sometimes difficult to discern
`Gene overexpression can also be used to assess function
`Research Briefing 7.1: Analysis of chromosome I of Caenorhabditis elegans by RNA interference
`7.2.3 More detailed studies of the activity of a protein coded by an unknown gene
`Directed mutagenesis can be used to probe gene function in detail
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`Technical Note 7. 1: Site-directed mutagenesis
`Reporter genes and immunocytochemistry can be used to locate where and when genes are expressed
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`G lobal Studies of Genome Activity
`7 .3
`7.3. 1 Studying the transcriptome
`The compos~ion of a transcriptome can be assayed by SAGE
`Using chip and microarray technology to study a transcriptome
`Studying the proteome
`Proteomics - methodology for characterizing the protein content of a cell
`Identifying proteins that interact w~h one another
`Protein interaction maps
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`7.3.2
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`7.4
`Co mparative Genomics
`7.4. 1 Comparative genomics as an aid to gene mapping
`7.4.2 Comparative genomics in the study of human disease genes
`Study Aids
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`PART 3
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`How Genomes Function
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`Chapter 8 Accessing the Genome
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`Inside the N ucleus
`8. 1
`8. 1. 1 The internal architecture of the eukaryotic nucleus
`Box 8. 1: Accessing the prokaryotic genome
`Technical Note 8. 1: Fluorescence recovery after photobleaching (FRAP)
`8. 1.2 Chromatin domains
`Functional domains are defined by insulators
`Some functional domains contain locus control regions
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`Chromatin Modifications and Genome Expression
`8.2
`8.2. 1 Activating the genome
`Histone modifications detenmine chromatin structure
`N ucleosome remodeling influences the expression of individual genes
`Box 8.2: C hromatin modification by the HMGN proteins
`Silencing the genome
`Histone deacetylation is one way of repressing gene expression
`Research Briefing 8.1: Discovery of the ma mmalian DNA methyltransferases
`Genome silencing by DNA methylation
`Methylation is involved in imprinting and X inactivation
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`8.2.2
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`Study Aids
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`Chapter 9 Assembly of the Transcription Initiation Complex
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`The Importance of DNA-binding Proteins
`9.1
`9. 1.1 Locating the positions of DNA-binding sites in a genome
`Gel retardation identifies DNA fragments that bind to proteins
`Protection assays pinpoint binding sites with greater accuracy
`M odification int erference ident ifies nucleotides central to prot ein binding
`Purifying a DNA-binding protein
`9. 1.2
`9. 1.3 Studying the structures of proteins and DNA-protein complexes
`X-ray crystallography has broad applications in structure detenminat ion
`NMR gives detailed structural information for small proteins
`9. 1.4 The special features of DNA-binding proteins
`The helix-turn-helix motif is present in prokaryotic and eukaryotic proteins
`Box 9.1 : RNA-b inding motifs
`Z inc fingers are common in eukaryotic proteins
`Other DNA-binding motifs
`Box 9.2: Can sequence specificity be predicted fro m t he structure of a recognitio n helix?
`9. 1.5 The interaction between DNA and its binding proteins
`Direct readout of the nucleotide sequence
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`The nucleotide sequence has a number o f indirect effects on helix structure
`Contacts between DNA and proteins
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`DNA-Protein Inte ract ions Du ring Tra nscription Initiation
`9.2
`9.2.1 RNA polymerases
`Box 9.3: Mitoch ondrial and chloroplast RNA polymerases
`9.2.2 Recognition sequences for transcription ini tiatio n
`Bacterial RNA polymerases bind to promoter sequences
`Eukaryotic promoters are more complex
`9.2.3 Assembly of the transcription initiation co mplex
`Transcription initiation in E. coli
`Transcript ion initiation with RNA polymerase II
`Box 9.4: Initiation of transcription in the archaea
`Transcription initiation with RNA polymerases I and Ill
`Research Briefing 9. 1: Similarities between TFHD and the hiscone core octamer
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`Regulation ofTranscription Initiation
`9.3
`9.3. I Strategies for controlling transcription initiation in bacteria
`Promoter structure determines the basal level of t ranscription initiation
`Regulatory control over bacterial transcription initiation
`Box 9.5: Cis and trans
`9.3.2 Control of transcription initiation in eukaryotes
`Activators of eukaryotic transcription initiation
`Contacts between activators and t he pre-initiation complex
`Repressors of eukaryotic transcription initiation
`Box 9.6: The modular structures of RNA polymerase II promoters
`Controlling the activities of activators and repressors
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`Study Aids
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`Chapter IO Synthesis and Processing of RNA
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`I 0. 1 Synthesis and Processing of mRNA
`I 0.1 . 1 Synthesis of bacterial mRNAs
`The elongation phase of bacterial transcription
`Termination of bacterial transcription
`Control over the choice between elongation and termination
`Research Briefing I 0. 1: The structure of the bacterial RNA polymerase
`Box I 0. 1: Antit'ermi nation during t he infect io n cycle of bac terio phage /._
`I 0. 1.2 Synthesis of eukaryotic mRNAs by RNA polymerase II
`Capping of RNA polymerase II transcripts occurs immediately after initiation
`Elongation of eukaryotic mRNAs
`Termination of mRNA synthesis is combined with polyadenylation
`lntron splicing
`_
`Conserved sequence motifs indicate the key sites in GU-AG intrans
`Outline of the splicing pathway for GU-AG intrans
`snRNAs and their associated proteins are the central components of the splicing apparatus
`Alternative splicing is common in many eukaryotes
`AU- AC intrans are similar to GU-AG intrans but require a different splicing apparatus
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`I 0.1.3
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`I 0.2 Synthesis and Processing of Non-coding RNAs
`I 0.2. 1 Transcript elongation and termination by RNA polymerases I and Ill
`I 0.2.2 Cutting events involved in processing of bacterial and eukaryotic pre-rRNA and pre-tRNA
`I 0.2.3 Intrans in eukaryotic pre-rRNA and pre-tRNA
`Eukaryotic pre-rRNA intrans are self-splicing
`Eukaryotic tRNA intrans are variable but all are spliced by the same mechanism
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`I 0.3 Processing of Pre-RNA by Chemical Modification
`I 0.3. 1 Chemical modification of pre-rRNAs
`Box I 0.2: Other types of incrnn
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`I 0.3.2 RNA editing
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`I 0 .4 Degradation of mRNAs
`Box I 0.3: More complex forms of RNA editing
`10.4. 1 Bacterial mRNAs are degraded in the 3'""5' direction
`I 0.4.2 Eukaryotes have more diverse mechanisms for RNA degradation
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`I 0.5 Transpo r t of RNA W ithin the Eukaryotic Cell
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`Study A id s
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`C hapter I I Synthesis and Processing of the Proteome
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`II.I
`II.I.I
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`11.1.2
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`I 1.2
`11.2.1
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`11.2.2
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`11.2.3
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`11.2.4
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`11.3
`11.3.1
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`11.3.2
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`11.3.3
`11.3.4
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`11.4
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`The Role of t RNA in Protein Synthesis
`Aminoacylation: the attachment of amino acids to tRNAs
`All tRNAs have a similar structure
`Aminoacyl-tRNA synthetases attach amino acids to tRNAs
`Codon-anticodon interactions: the attachment of tRNAs to mRNA
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`The Role of the Ribosome in Protein Synthesis
`Ribosome structure
`Ultracentrifugation was used to measure the sizes of ribosomes and their components
`Probing the fine structure of the ribosome
`Initiation of translation
`Initiation in bacteria requires an internal ribosome binding site
`Initiation in eukaryotes is mediated by the cap structure and poly(A) tail
`Initiation of eukaryotic translation without scanning
`Regulation of translation initiation
`Elongation of translation
`Elongation in bacteria and eukaryotes
`Frameshifting and other unusual events during elongation
`Research Briefing 11 . 1: Peptidyl transferase is a ribozyme
`Termination of translation
`Box I I. I: Translation in the archaea
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`Post-translational Processing of Proteins
`Protein folding
`Not all proteins fold spontaneously in the test tube
`In cells, folding is aided by molecular chaperones
`Processing by proteolytic cleavage
`Cleavage of the ends of polypeptides
`Proteolytic processing of polyproteins
`Processing by chemical modification
`lnteins
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`Protein Degradation
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`Study Aids
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`12. 1
`12. 1.I
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`Chapter 12 Regulation of Genome Activity
`Transient Changes in Genome Activity
`Signal transmission by import of the extracellular signaling compound
`Lactoferrin is an extracellular signaling protein which acts as a transcription activator
`Some imported signaling compounds directly influence the activity of pre-existing protein factors
`Some imported signaling compounds influence genome activity indirectly
`Signal transmission mediated by cell surface receptors
`Signal transduction with one step between receptor and genome
`Signal transduction with many steps between receptor and genome
`Signal transduction via second messengers
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`12.1.2
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`12.2 Permanent and Semipermanent Changes in Genome Activity
`12.2.1 Genome rearrangements
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`Yeast mating types are determined by gene conversion events
`Genome reammgements are responsible for immunoglobulin and T-cell receptor d1versrt1es
`12.2.2 Changes in chromatin structure
`12.2.3 Genome regulation by feedback loops
`Research Briefing 12 .1 : Unraveling a signai transduction pathway
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`12.3 Regulation of Geno m e A ctivity During Development
`12.3 . 1 Sporulation in Bacillus
`Sporulation involves coordinated activities in tw o distinct cell types
`Special cr subunits control genome activity during sporulation
`12.3.2 Vulva development in Caenorhabditis elegans
`C. elegans is a model for multicellular eukaryotic development
`Determination of cell fate during development of the C. elegans vulva
`Research Briefing 12.2: T11e link between genome replicat ion and sporulation in Bacillus
`12.3.3 Development in Drosophila melanogaster
`Maternal genes establish protein gradients in the Drosqphila embryo
`A cascade of gene expression converts positional information into a segmentation pattern
`Box 12. I: T he genetic basis of flower development
`Segment identity is determined by the homeotic selector genes
`Homeotic selector genes are universal features of higher eukaryotic development
`
`Study Aids
`
`PART4
`
`How Genomes Replicate and Evolve
`
`Chapt e r I 3 Genome Replication
`
`13. 1
`13. 1.1
`
`13. 1.2
`13.1.3
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`13.2
`13.2. 1
`
`13.2.2
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`13.2.3
`
`13.2.4
`
`13.3
`13.3. 1
`
`13.3.2
`
`The Topo logical Proble m
`Experimental proof for the Watson-Crick scheme for DNA replication
`The Meselson-Stahl experiment
`DNA topoisomerases provide a solution to the topological problem
`Variations on the semiconservative theme
`
`The Replication Process
`Initiation of genome replication
`Initiation at the E coli origin of replication
`Origins of replication in yeast have been clearly defined
`Replication origins in higher eukaryotes have been less easy to identify
`The elongation phase of replication
`The DNA polymerases of bacteria and eukaryotes
`Discontinuous strand synthesis and the priming problem
`Events at the bacterial replication fork
`The eukaryotic replication fork: variations on the bacterial theme
`Termination of replication
`Replication of the E. coli genome terminates within a defined region
`Little is known about termination of replication in eukaryotes
`Box 13. 1: Genome replication in the archaea
`Maintaining the ends of a linear DNA molecule
`Telomeric DNA is synthesized by the telomerase enzyme
`Telomere length is implicated in senescence and cancer
`Box 13.2: Telomeres in Drosopl,ilo
`
`Regulat ion o f Eukaryotic Genome Replication
`Coordination of genome replication and cell division
`Establishment of the pre-replication complex enables genome replication to commence
`Research Briefing 13. 1: Replication of the yeast genome
`Regulation of pre-RC assembly
`Control within S phase
`Early and late replicat ion origins
`Checkpoints within S phase
`
`Study A ids
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`

`Cha pter 14 Mutation, Repair and Recombination
`
`14. 1
`14. 1.1
`
`14.1.2
`
`14. 1.3
`
`14.2
`14.2.1
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`14.2.2
`
`14.2.3
`14.2.4
`14.2.5
`14.2.6
`
`14.3
`14.3.1
`
`14.3.2
`
`14.3.3
`
`Box 14.1: Terminology for describing point muta ti ons
`Mutations
`The causes of mutations
`Errors in replication are a source of point mutations
`Replication errors can also lead to insertion arid deletion mutations
`Mutations are also caused by chemical and physical mutagens
`The effects of mutations
`The effects of mutations on genomes
`lt.:chnical Note 14.1: Mut.1tion detection
`The effects of mutations on multicellular organisms
`The effects of mutations on microorganisms
`Hypermutation and the possibility of programmed mutations
`
`DNA Repair
`Direct repair systems fill in nicks and correct some types of nucleotide modification
`Research Briefing 14.1: Programmed mutations?
`Excision repair
`Base excision repairs many types of damaged nucleotide
`Nucleotide excision repair is used to correct more extensive types of damage
`Mismatch repair: correcting errors of replication
`Repair of double-stranded DNA breaks
`Bypassing DNA damage during genome replication
`Defects in DNA repair underlie human diseases, including cancers
`
`Recombination
`Homologous recombination
`The Holliday model for homologous recombination
`Proteins involved in homologous recombination in £ coli
`The double-strand break model for recombination in yeast
`Box 14.2: The RecE and Reef recombination pathways of Escherichia coli
`Site-specific recombination
`Integration of 1,. DNA into the E. coli genome involves site-specific recombination
`Transposition
`Box 14.3: DNA methylation and transposition
`Replicative and conservative transposition of DNA transposons
`Transposition of retroelements
`
`Study Aids
`
`Chapter I 5 How Genomes Evolve
`
`IS. I
`IS. I.I
`
`Genomes: the First IO Billion Years
`The origins of genomes
`The first biochemical systems were centered on RNA
`The first DNA genomes
`How unique is life?
`
`15.2
`15.2.1
`
`15.2.2
`
`15.3
`15.3.I
`
`15.3.2
`
`A cq uisitio n of N ew Genes
`Acquisition of new genes by gene duplication
`Whole-genome duplications can result in sudden expansions in gene number
`Research Briefing 15. 1: Segmental duplic:ltions in the yeast and human geno mes
`Duplications of individual genes and groups of genes have occurred frequ ently in the past
`Box 15.1: Gene duplication and genetic redundancy
`Genome evolution also involves rearrangement of existing genes
`Acquisition of new genes from other species
`
`N on-coding DNA and Genome Evolution
`Transposable elements and genome evolution
`Box 15.2: The origin of a microsatcllitc
`The origins of intrans
`
`CONTENTS xv
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`xvi CONTENTS
`
`'lntrons early' and 'introns late': two competing hypotheses
`The current evidence disproves neither hypothesis
`_ rvlc Gf non-coding DNA
`
`15 ,j
`
`·1;i '.':
`
`..... ~r.:1..1n G enome:the Last S M illion Years
`
`Molecular Phylogenetics
`I ,:,, 1 ~!,~ O rigins of Molecular Phylogenetics
`..... ,.
`:,. ! : ehenccics and c\Jdistics
`
`i!· -, Th:: Re const ruction of DN A-based P hylogenetic Trees
`16.2. 1 The key features of DNA-based phylogenetic trees
`Gene trees are not the same as species trees
`de:,. ·
`,
`... \
`·1inotogy for molec ular phylogenetics
`16.2.2 Tree reconstruction
`Sequence alignment is the essential preliminary to tree reconstruction
`Converting alignment data into a phylogenetic tree
`Technical Note 16.1: Phylogenetic analysis
`Assessing the accuracy of a reconstructed tree
`Molecular clocks enable the time of divergence of ancestral sequences to be estimated
`
`16.3 The Applications of Molecular Phylogenetics
`16.3. 1 Examples of the use of phylogenetic trees
`DNA phylogenetics has clarified the evolutionary relationships between humans and other primates
`The origins of AIDS
`16.3.2 Molecular phylogenetics as a tool in the study of human prehistory
`lntraspecific studies require highly variable genetic loci
`The origins of modern humans - out of Africa or not?
`Box 16.3: Genes in populations
`The patterns of more recent migrat ions into Europe are also controversial
`Research Briefing 16. 1: Neandertal DNA
`Prehistoric human migrations into the New World
`
`Study Aids
`
`Appendix
`
`Glossary
`Index
`
`Keeping up to Date
`
`Keeping up to Date by Reading the Literature
`
`Keeping up to Date using the Internet
`
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`__ ..._ __ ... ,.
`
`~-
`
`~ i ,
`
`CHAPTER
`
`Studying DNA
`
`Chapter Contents
`
`4.1 Enzymes for DNA Manipulation
`4.1.1 D NA polymerases
`4.1.2 Nucleases
`4.1.3 D NA ligases
`4.1.4 End-modification enzymes
`
`4.2 DNA Cloning
`4.2.1 Cloning vectors and the way
`they are used
`
`4.3 The Polymerase Chain Reaction
`(PCR)
`4.3.1 Carrying out a PCR
`4.3.2 The applications of PCR
`
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`98
`102
`105
`107
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`108
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`109
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`96 CHAPTER 4 • STUDYING DNA
`
`Learning outcomes
`
`When you have read Chapter 4, you should be able to:
`
`• Gi~e outline descriptions of the events involved in DNA cloning and the polymerase chain
`reaction (PCR), and state the applications and limitations of these techniques
`
`• Describe the activities and main applications of the different types of enzyme used in
`· recombinant DNA research
`
`•
`
`Identify the important features of DNA polymerases and distinguish between the various
`DNA polymerases used in genomics research
`
`• Describe, with examples, the way that restriction endonucleases cut DNA and explain how
`the results of a restriction digest are examined
`
`• Distinguish between blunt- and sticky-end ligation and explain how the efficiency of blunt-end
`ligation can be increased
`
`• Give details of the key features of plasmid cloning vectors and describe how these vectors are
`used in cloning experiments, using pBR322 and pUCB as examples
`
`• Describe how bacteriophage '),,, vectors are used to clone DNA
`
`• Give examples of vectors used to clone long pieces of DNA, and evaluate the strengths and
`weaknesses of each type
`
`•
`
`Summarize how DNA is cloned in yeast, animals and plants
`
`• Describe how a PCR is performed, paying particular attention to the importance of the
`primers and the temperatures used during the thermal cycling
`
`THE TOOLKIT OF TECHNIQUES used by molecular biologists
`to study DNA molecules was assembled during the
`1970s and 1980s. Before then, the only way in which
`individual genes could be studied was by classical
`genetics, using the procedures that we will examine in
`Chapter 5. Classical genetics is a powerful approach to
`gene analysis and many of the fundamental discoveries
`in molecular biology were made in this way. The
`operon theory proposed by Jacob and Monad in 1961
`(Section 9.3.1), which describes how the expression of
`some bacterial genes is regulated, was perhaps the
`most heroic achievement of this era of genetics. But the
`classical approach
`is limited because it does not
`involve the direct examination of genes, information on
`gene structure and activity being inferred from the bio(cid:173)
`logical characteristics of the organism being studied. By
`the late 1960s these indirect methods had become
`insufficient for answering the more detailed questions
`that molecular biologists had begun to ask about the
`expression pathways of individual genes. These ques(cid:173)
`tions could only be ·addressed by examining directly
`the segments of DNA containing the genes of interest.
`
`This was not possible using the current technology, so
`a new set of techniques had to be invented.
`The development of these new techniques was stimu(cid:173)
`lated by breakthroughs in biochemical research which, in
`the early 1970s, provided molecular biologists with
`enzymes that could be used to manipulate DNA mole(cid:173)
`cules in the test tube. These enzymes occur naturally in
`living cells and are involved in processes such as DNA
`replication, repair and recombination (see Chapters 13
`and 14). In order to determine the functions of these
`enzymes, many of