Executive Editor: Gary Carlson
Editor-in-Chief: John Challice
President: Paul F. Corey
Assistant Vice President of Product...
instant access to key research in this field. The unique user-interface
allows you to view the site in three different for...
Outline
Part 1 Genes Part 5 The Nucleus
1 Genes are DNA
2 The interrupted gene
3 The content of the genome
4 Clusters and ...
Contents
Part 1 Genes
1 Genes are DNA
1.1 Introduction
1.2 DNA is the genetic material of bacteria
1.3 DNA is the genetic ...
3.5 Why are genomes so large? 56
3.6 Eukaryotic genomes contain both nonrepetitive and repetitive DNA sequences 57
3.7 Bac...
6 Protein synthesis
6.1 Introduction 135
6.2 Protein synthesis occurs by initiation, elongation, and termination 136
6.3 S...
8.17 Post-translational membrane insertion depends on leader sequences 214
8.18 A hierarchy of sequences determines locati...
10.12 The repressor monomer has several domains 290
10.13 Repressor is a tetramer made of two dimers 291
10.14 DNA-binding...
Part 4 DNA
13 The replicon
13.1 Introduction 353
13.2 Replicons can be linear or circular 355
13.3 Origins can be mapped b...
15.6 The synaptonemal complex forms after double-strand breaks 426
15.7 Pairing and synaptonemal complex formation are ind...
18 Rearrangement of DNA
18.1 Introduction 513
18.2 The mating pathway is triggered by pheromone-receptor interactions 514
...
20.12 Are transcribed genes organized in nucleosomes? 587
20.13 Histone octamers are displaced by transcription 588
20.14 ...
23.8 Deacetylases are associated with repressors 666
23.9 Methylation of histones and DNA is connected 667
23.10 Chromatin...
26.3 Immunoglobulin genes are assembled from their parts in lymphocytes 754
26.4 Light chains are assembled by a single re...
28.18 Activation of a pathway can produce different results 834
28.19 Cyclic AMP and activation of CREB 835
28.20 The JAK-...
30.21 p53 is a DNA-binding protein 919
30.22 p53 is controlled by other tumor suppressors and oncogenes 921
30.23 p53 is a...
GENES is continuously updated on the web site, www.ergito.com
with revisions posted weekly. This allows readers to check f...
Chapter 1
Genes are DNA
1.1 Introduction
The hereditary nature of every living organism is defined by its
genome, which co...
Each chromosome consists of a linear array of genes. Each gene re-
sides at a particular location on the chromosome. This ...
1.2 DNA is the genetic material of bacteria
The idea that genetic material is nucleic acid had its roots in the
discovery ...
I
bacterium E. coli. When phage particles are added to bacteria, they ad-
sorb to the outside surface, some material enter...
ribonucleic acid (RNA), as the genetic material. The general principle
of the nature of the genetic material, then, is tha...
1.6 DNA is a double helix
Figure 1.8 The double helix maintains a
constant width because purines always
face pyrimidines i...
terms of a spiral staircase: the base pairs form the treads, as illustrated
schematically in Figure 1.9. Proceeding along ...
medium containing a suitable isotope (such as 15
N), its strands can be
distinguished from those that are synthesized when...
Endonucleases cut individual bonds within RNA or DNA molecules,
generating discrete fragments. Some DNAases cleave both st...
authentic protein structure; it is a general term implying that the natural
conformation of a macromolecule has been conve...
Mutations provide decisive evidence that DNA is the genetic ma-
terial. When a change in the sequence of DNA causes an alt...
• Chemical modification of DNA directly changes one base into a dif-
ferent base.
• A malfunction during the replication o...
1.12 The effects of mutations can be reversed
Key Concepts
• Forward mutations inactivate a gene, and back mutations (or
r...
any part of it can abolish its function. As a result, susceptibility to mu-
tation is roughly proportional to the size of ...
Figure 1.25 shows that the consequences of deamination are different
for 5-methylcytosine and cytosine. Deaminating the (r...
The first systematic attempt to associate genes with enzymes
showed that each stage in a metabolic pathway is catalyzed by...
ferent genes whose proteins are involved in the same function. The
complementation test is used to determine whether two m...
1.17 Mutations may cause loss-of-function or
gain-of-function
Key Concepts
• Recessive mutations are due to loss-of-functi...
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  1. 1. Executive Editor: Gary Carlson Editor-in-Chief: John Challice President: Paul F. Corey Assistant Vice President of Production and Manufacturing: David Riccardi Manager of Electronic Composition: Jim Sullivan Executive Managing Editor: Kathleen Schiaparelli Editorial Assistant: Susan Zeigler Assistant Managing Editor, Science Media: Nicole Bush Media Editor: Andrew Stall Assistant Editor: Chrissy Dudonis Senior Marketing Manager: Shari Mcffert Art Director: John Christiana Book Design: Bang Wong (Virtual Text) Manufacturing Buyer: Alan Fischer Manufacturing Manager: Trudy Pisciotti Marketing Assistant: Juliana Tarris Director of Creative Services: Paul Belfanti Cover Designer: Bruce Kenselaar Cover Credit: High Density Liquid Crystalline DNA by Michael W. Davidson and The Florida State University (National High Magnetic Field Laboratory) © 2004 by Benjamin Lewin Published by Pearson Prentice Hall Pearson Education, Inc. Upper Saddle River, NT 07458 All rights reserved. No part of this book may be reproduced, in any form or by any means, without permission in writing from the publisher. Pearson Prentice Hall® is a trademark of Pearson Education, Inc. If you purchased this book within the United States or Canada you should be aware that it has been wrongfully imported without the approval of the Publisher or the Author. Printed in the United States of America ] 0 9 8 7 6 5 4 3 2 ISBN D-13-lE3flHb-4 Pearson Education LTD., London Pearson Education Australia PTY, Limited, Sydney Pearson Education Singapore, Pte. Ltd Pearson Education North Asia Ltd, Hong Kong Pearson Education Canada, Ltd., Toronto Pearson Educacion de Mexico, S.A. de C.V. Pearson Education—Japan, Tokyo Pearson Education Malaysia, Pte. Ltd Pearson Education Inc., Upper Saddle River, New Jersey By Book_Crazy [IND]
  2. 2. instant access to key research in this field. The unique user-interface allows you to view the site in three different formats, highlighting text, images or a combination of both, to best support your teaching style. Instructor's Resource Manual (0-13-144944-3) Test Item File (0-13-144945-1) Transparency Package (0-13-144946-X) For the Student: Student Study Companion: This study tool provides students with the resources to review fundamental concepts from the text through prac- tice questions and exercises. Additional study aids help students-to study more effectively. Website with E-Book (www.prenhall.com/lewin) This powerful website contains an online version of the text, supported by weekly updates to maintain currency on key topics. Links connect the student directly to the original source material for immediate access to key articles wherever possible. The unique user-interface allows students to view the site in three different formats, highlighting text, images or a combi- nation of both, to best support their learning style. v j PREFACE By Book_Crazy [IND]
  3. 3. Outline Part 1 Genes Part 5 The Nucleus 1 Genes are DNA 2 The interrupted gene 3 The content of the genome 4 Clusters and repeats Part 2 Proteins 5 Messenger RNA 6 Protein synthesis 7 Using the genetic code 8 Protein localization Part 3 Gene expression 9 Transcription 10 The operon 11 Regulatory circuits 12 Phage strategies Part 4 DNA 13 The replicon 14 DNA replication 15 Recombination and repair 16 Transposons 17 Retroviruses and retroposons 18 Rearrangement of DNA 1 33 51 85 113 135 167 195 241 279 301 329 353 387 419 467 493 513 19 Chromosomes 545 20 Nucfeosomes 571 21 Promoters and enhancers 597 22 Activating transcription 631 23 Controlling chromatin structure 657 24 RNA splicing and processing 697 25 Catalytic RNA 731 26 Immune diversity 751 Part 6 Cells 27 Protein trafficking 787 28 Signal transduction 811 29 Cell cycle and growth regulation 843 30 Oncogenes and cancer 889 31 Gradients, cascades, and signaling pathways 939 Glossary 981 Index 1003 OUTLINE VII By Book_Crazy [IND]
  4. 4. Contents Part 1 Genes 1 Genes are DNA 1.1 Introduction 1.2 DNA is the genetic material of bacteria 1.3 DNA is the genetic material of viruses 1.4 DNA is the genetic material of animal cells 1.5 Polynucleotide chains have nitrogenous bases linked to a sugar-phosphate backbone 1.6 DNA is a double helix 1.7 DNA replication is semiconservative 1.8 DNA strands separate at the replication fork 1.9 Nucleic acids hybridize by base pairing 1.10 Mutations change the sequence of DNA 1.1 1 Mutations may affect single base pairs or longer sequences 1.12 The effects of mutations can be reversed 1.13 Mutations are concentrated at hotspots 1.14 Many hotspots result from modified bases 1.15 A gene codes for a single polypeptide 1.16 Mutations in the same gene cannot complement 1.17 Mutations may cause loss-of-function or gain-of-function 1.18 A locus may have many different mutant alleles 1.19 A locus may have more than one wild-type allele 1.20 Recombination occurs by physical exchange of DNA 1.21 The genetic code is triplet 1.22 Every sequence has three possible reading frames 1.23 Prokaryotic genes are colinear with their proteins 1.24 Several processes are required to express the protein product of a gene 1.25 Proteins are frans-acting but sites on DNA are c/s-acting 1.26 Genetic information can be provided by DNA or RNA 1.27 Some hereditary agents are extremely small 1.28 Summary 1 3 3 4 5 6 7 8 9 10 1 1 13 13 14 15 16 18 18 19 20 21 23 24 25 26 27 29 30 2 The interrupted gene 2.1 Introduction 2.2 An interrupted gene consists of exons and introns 2.3 Restriction endonucleases are a key tool in mapping DNA 2.4 Organization of interrupted genes may be conserved 2.5 Exon sequences are conserved but introns vary 2.6 Genes can be isolated by the conservation of exons 2.7 Genes show a wide distribution of sizes 2.8 Some DNA sequences code for more than one protein 2.9 How did interrupted genes evolve? 2.10 Some exons can be equated with protein functions 2.11 The members of a gene family have a common organization 2.12 Is all genetic information contained in DNA? 2.13 Summary 33 34 35 36 37 38 40 41 43 45 46 48 49 3 The content of the genome 3.1 Introduction 3.2 Genomes can be mapped by linkage, restriction cleavage, or DNA sequence 3.3 Individual genomes show extensive variation 3.4 RFLPs and SNPs can be used for genetic mapping 51 52 53 54 CONTENTS IX By Book_Crazy [IND]
  5. 5. 3.5 Why are genomes so large? 56 3.6 Eukaryotic genomes contain both nonrepetitive and repetitive DNA sequences 57 3.7 Bacterial gene numbers range over an order of magnitude 58 3.8 Total gene number is known for several eukaryotes 60 3.9 How many different types of genes are there? 61 3.10 The conservation of genome organization helps to identify genes 63 3.11 The human genome has fewer genes than expected 65 3.12 How are genes and other sequences distributed in the genome? 67 3.13 More complex species evolve by adding new gene functions 68 3.14 How many genes are essential? 69 3.15 Genes are expressed at widely differing levels 72 3.16 How many genes are expressed? 73 3.17 Expressed gene number can be measured en masse 74 3.18 Organelles have DNA - 75 3.19 Organelle genomes are circular DNAs that code for organelle proteins 76 3.20 Mitochondrial DNA organization is variable 77 3.21 Mitochondria evolved by endosymbiosis 78 3.22 The chloroplast genome codes for many proteins and RNAs 79 3.23 Summary 80 4 Clusters and repeats 4.1 Introduction 85 4.2 Gene duplication is a major force in evolution 86 4.3 Globin clusters are formed by duplication and divergence 87 4.4 Sequence divergence is the basis for the evolutionary clock 89 4.5 The rate of neutral substitution can be measured from divergence of repeated sequences 92 4.6 Pseudogenes are dead ends of evolution 93 4.7 Unequal crossing-over rearranges gene clusters 95 4.8 Genes for rRNA form tandem repeats 98 4.9 The repeated genes for rRNA maintain constant sequence 99 4.10 Crossover fixation could maintain identical repeats 100 4.1 1 Satellite DNAs often lie in heterochromatin 103 4.12 Arthropod satellites have very short identical repeats 105 4.13 Mammalian satellites consist of hierarchical repeats 106 4.14 Minisatellites are useful for genetic mapping 109 4.1 5 Summary 111 Part 2 Proteins 5 Messenger RNA 5.1 Introduction 113 5.2 mRNA is produced by transcription and is translated 1 14 5.3 Transfer RNA forms a cloverleaf 114 5.4 The acceptor stem and anticodon are at ends of the tertiary structure 1 16 5.5 Messenger RNA is translated by ribosomes 11 7 5.6 Many ribosomes bind to one mRNA 118 5.7 The life cycle of bacterial messenger RNA 1 1 9 5.8 Eukaryotic mRNA is modified during or after its transcription 121 5.9 The 5' end of eukaryotic mRNA is capped 122 5.10 The 3' terminus is polyadenylated 123 5.11 Bacterial mRNA degradation involves multiple enzymes 124 5.12 mRNA stability depends on its structure and sequence 125 5.13 mRNA degradation involves multiple activities 126 5.14 Nonsense mutations trigger a surveillance system 127 5.15 Eukaryotic RNAs are transported 128 5.16 mRNA can be specifically localized 130 5.17 Summary 131 CONTENTS By Book_Crazy [IND]
  6. 6. 6 Protein synthesis 6.1 Introduction 135 6.2 Protein synthesis occurs by initiation, elongation, and termination 136 6.3 Special mechanisms control the accuracy of protein synthesis 138 6.4 Initiation in bacteria needs 30S subunits and accessory factors 139 6.5 A special initiator tRNA starts the polypeptide chain 140 6.6 Use of fMet-tFSNAf is controlled by IF-2 and the ribosome 141 6.7 Initiation involves base pairing between mRNA and rRNA 142 6.8 Small subunits scan for initiation sites on eukaryotic mRNA 144 6.9 Eukaryotes use a complex of many initiation factors 146 6.10 Elongation factor Tu loads aminoacyl-tRNA into the A site 148 6.11 The polypeptide chain is transferred to aminoacyl-tRNA 149 6.12 Translocation moves the ribosome 150 6.13 Elongation factors bind alternately to the ribosome 151 6.14 Three codons terminate protein synthesis 152 6.15 Termination codons are recognized by protein factors 153 6.16 Ribosomal RNA pervades both ribosomal subunits 155 6.17 Ribosomes have several active centers 157 6.18 16S rRNA plays an active role in protein synthesis 159 6.19 23S rRNA has peptidyl transferase activity 161 6.20 Summary 162 7 Using the genetic code 7.1 Introduction 167 7.2 Codon-anticodon recognition involves wobbling 169 7.3 tRNAs are processed from longer precursors 170 7.4 tRNA contains modified bases 171 7.5 Modified bases affect anticodon-codon pairing 173 7.6 There are sporadic alterations of the universal code 174 7.7 Novel amino acids can be inserted at certain stop codons 176 7.8 tRNAs are charged with amino acids by synthetases 177 7.9 Aminoacyl-tRNA synthetases fall into two groups 178 7.10 Synthetases use proofreading to improve accuracy 180 7.11 Suppressor tRNAs have mutated anticodons that read new codons 182 7.12 There are nonsense suppressors for each termination codon 183 7.13 Suppressors may compete with wild-type reading of the code 184 7.14 The ribosome influences the accuracy of translation 185 7.15 Recoding changes codon meanings 188 7.16 Frameshifting occurs at slippery sequences 189 7.17 Bypassing involves ribosome movement 190 7.18 Summary 191 8 Protein localization 8.1 Introduction 195 8.2 Passage across a membrane requires a special apparatus 196 8.3 Protein translocation may be post-translational or co-translational 197 8.4 Chaperones may be required for protein folding 198 8.5 Chaperones are needed by newly synthesized and by denatured proteins 199 8.6 The Hsp70 family is ubiquitous 201 8.7 Hsp60/GroEL forms an oligomeric ring structure 202 8.8 Signal sequences initiate translocation 203 8.9 The signal sequence interacts with the SRP 205 8.10 The SRP interacts with the SRP receptor 206 8.11 The translocon forms a pore 207 8.12 Translocation requires insertion into the translocon and (sometimes) a ratchet in the ER 209 8.13 Reverse translocation sends proteins to the cytosol for degradation 210 8.14 Proteins reside in membranes by means of hydrophobic regions 211 8.15 Anchor sequences determine protein orientation 212 8.16 How do proteins insert into membranes? 213 CONTENTS XI By Book_Crazy [IND]
  7. 7. 8.17 Post-translational membrane insertion depends on leader sequences 214 8.18 A hierarchy of sequences determines location within organelles 215 8.19 Inner and outer mitochondrial membranes have different translocons 217 8.20 Peroxisomes employ another type of translocation system 219 8.21 Bacteria use both co-translational and post-translational translocation 220 8.22 The Sec system transports proteins into and through the inner membrane 221 8.23 Sec-independent translation systems in E. coll 222 8.24 Pores are used for nuclear import and export 223 8.25 Nuclear pores are large symmetrical structures 224 8.26 The nuclear pore is a size-dependent sieve for smaller material 225 8.27 Proteins require signals to be transported through the pore 226 8.28 Transport receptors carry cargo proteins through the pore 227 8.29 Ran controls the direction of transport 228 8.30 RNA is exported by several systems 230 8.31 Ubiquitination targets proteins for degradation 231 8.32 The proteasome is a large machine that degrades ubiquitinated proteins 232 8.33 Summary 234 Part 3 Gene expression 9 Transcription 9.1 Introduction 241 9.2 Transcription occurs by base pairing in a "bubble" of unpaired DNA 242 9.3 The transcription reaction has three stages 243 9.4 Phage T7 RNA polymerase is a useful model system 244 9.5 A model for enzyme movement is suggested by the crystal structure 245 9.6 Bacterial RNA polymerase consists of multiple subunits 246 9.7 RNA polymerase consists of the core enzyme and sigma factor 248 9.8 The association with sigma factor changes at initiation 249 9.9 A stalled RNA polymerase can restart 250 9.10 How does RNA polymerase find promoter sequences? 251 9.1 1 Sigma factor controls binding to DNA 252 9.12 Promoter recognition depends on consensus sequences 253 9.13 Promoter efficiencies can be increased or decreased by mutation 255 9.14 RNA polymerase binds to one face of DNA 256 9.15 Supercoiling is an important feature of transcription 258 9.16 Substitution of sigma factors may control initiation 259 9.17 Sigma factors directly contact DNA 261 9.18 Sigma factors may be organized into cascades 263 9.19 Sporulation is controlled by sigma factors 264 9.20 Bacterial RNA polymerase terminates at discrete sites 266 9.21 There are two types of terminators in E. coli 267 9.22 How does rho factor work? 268 9.23 Antitermination is a regulatory event 270 9.24 Antitermination requires sites that are independent of the terminators 271 9.25 Termination and anti-termination factors interact with RNA polymerase 272 9.26 Summary 274 10 The operon 10.1 Introduction 279 10.2 Regulation can be negative or positive 280 10.3 Structural gene clusters are coordinately controlled 281 10.4 The lac genes are controlled by a repressor 282 10.5 The lac operon can be induced 283 10.6 Repressor is controlled by a small molecule inducer 284 10.7 c/s-acting constitutive mutations identify the operator 286 10.8 frans-acting mutations identify the regulator gene 287 10.9 Multimeric proteins have special genetic properties 288 10.10 Repressor protein binds to the operator 288 10.11 Binding of inducer releases repressor from the operator 289 XII CONTENTS By Book_Crazy [IND]
  8. 8. 10.12 The repressor monomer has several domains 290 10.13 Repressor is a tetramer made of two dimers 291 10.14 DNA-binding is regulated by an allosteric change in conformation 291 10.15 Mutant phenotypes correlate with the domain structure 292 10.16 Repressor binds to three operators and interacts with RNA polymerase 293 10.17 Repressor is always bound to DNA 294 10.18 The operator competes with low-affinity sites to bind repressor 295 10.19 Repression can occur at multiple loci 297 10.20 Summary 298 11 Regulatory circuits 11.1 Introduction 301 11.2 Distinguishing positive and negative control 302 11.3 Glucose repression controls use of carbon sources 304 1 1.4 Cyclic AMP is an inducer that activates CRP to act at many operons 305 11.5 CRP functions in different ways in different target operons 305 11.6 CRP bends DNA 307 11.7 The stringent response produces (p)ppGpp 308 11.8 (p)ppGpp is produced by the ribosome 309 11.9 ppGpp has many effects 310 11.10 Translation can be regulated 311 11.11 r-protein synthesis is controlled by autogenous regulation 312 11.12 Phage T4 p32 is controlled by an autogenous circuit 31 3 11.13 Autogenous regulation is often used to control synthesis of macromolecular assemblies 314 11.14 Alternative secondary structures control attenuation 315 11.15 Termination of B. subtilis trp genes is controlled by tryptophan and by tRNATrp 316 11.16 The E. coli tryptophan operon is controlled by attenuation 316 11.17 Attenuation can be controlled by translation 31 8 11.18 Antisense RNA can be used to inactivate gene expression 319 11.19 Small RNA molecules can regulate translation 320 11.20 Bacteria contain regulator RNAs 321 11.21 MicroRNAs are regulators in many eukaryotes 322 11.22 RNA interference is related to gene silencing 323 1 1.23 Summary 325 1 2 Phage strategies 12.1 Introduction 329 12.2 Lytic development is divided into two periods 330 12.3 Lytic development is controlled by a cascade 331 12.4 Two types of regulatory event control the lytic cascade 332 12.5 The T7 and T4 genomes show functional clustering 333 12.6 Lambda immediate early and delayed early genes are needed for both iysogeny and the lytic cycle 334 12.7 The lytic cycle depends on antitermination 335 12.8 Lysogeny is maintained by repressor protein 336 12.9 Repressor maintains an autogenous circuit 337 12.10 The repressor and its operators define the immunity region 338 12.11 The DNA-binding form of repressor is a dimer 339 12.12 Repressor uses a helix-turn-helix motif to bind DNA 340 12.13 The recognition helix determines specificity for DNA 340 12.14 Repressor dimers bind cooperatively to the operator 342 12.15 Repressor at OR2 interacts with RNA polymerase at PRM 343 12.16 The ell and c///genes are needed to establish lysogeny 344 12.17 A poor promoter requires ell protein t 345 12.18 Lysogeny requires several events 346 12.19 The cro repressor is needed for lytic infection 347 12.20 What determines the balance between lysogeny and the lytic cycle? 349 12.21 Summary 350 CONTENTS XIII By Book_Crazy [IND]
  9. 9. Part 4 DNA 13 The replicon 13.1 Introduction 353 13.2 Replicons can be linear or circular 355 13.3 Origins can be mapped by autoradiography and electrophoresis 355 13.4 The bacterial genome is a single circular replicon 356 13.5 Each eukaryotic chromosome contains many replicons 358 13.6 Replication origins can be isolated in yeast 359 13.7 D loops maintain mitochondrial origins 361 13.8 The ends of linear DNA are a problem for replication 362 13.9 Terminal proteins enable initiation at the ends of viral DNAs 363 13.10 Rolling circles produce multimers of a replicon 364 1 3.1 1 Rolling circles are used to replicate phage genomes 364 13.12 The F plasmid is transferred by conjugation between bacteria 366 13.13 Conjugation transfers single-stranded DNA 367 13.14 Replication is connected to the cell cycle 368 13.15 The septum divides a bacterium into progeny each containing a chromosome 370 13.16 Mutations in division or segregation affect cell shape 371 13.17 FtsZ is necessary for septum formation 372 13.18 min genes regulate the location of the septum 373 13.19 Chromosomal segregation may require site-specific recombination 374 13.20 Partitioning involves separation of the chromosomes 375 13.21 Single-copy plasmids have a partitioning system 377 13.22 Plasmid incompatibility is determined by the replicon 379 13.23 The ColEI compatibility system is controlled by an RNA regulator 380 13.24 How do mitochondria replicate and segregate? 382 13.25 Summary 383 14 DNA replication 14.1 Introduction 387 14.2 DNA polymerases are the enzymes that make DNA 388 14.3 DNA polymerases have various nuclease activities 389 14.4 DNA polymerases control the fidelity of replication 390 14.5 DNA polymerases have a common structure 391 14.6 DNA synthesis is semidiscontinuous 392 14.7 The <[>X model system shows how single-stranded DNA is generated for replication 393 14.8 Priming is required to start DNA synthesis 394 14.9 Coordinating synthesis of the lagging and leading strands 396 14.10 DNA polymerase holoenzyme has 3 subcomplexes 397 14.11 The clamp controls association of core enzyme with DNA 398 14.12 Okazaki fragments are linked by ligase 399 14.13 Separate eukaryotic DNA polymerases undertake initiation and elongation 400 14.14 Phage T4 provides its own replication apparatus 402 14.15 Creating the replication forks at an origin 404 14.16 Common events in priming replication at the origin 405 14.17 The primosome is needed to restart replication 407 14.18 Does methylation at the origin regulate initiation? 408 14.19 Origins may be sequestered after replication 409 14.20 Licensing factor controls eukaryotic rereplication 41 1 14.21 Licensing factor consists of MCM proteins 412 14.22 Summary 413 15 Recombination and repair 15.1 Introduction 419 15.2 Homologous recombination occurs between synapsed chromosomes 420 15.3 Breakage and reunion involves heteroduplex DNA 422 15.4 Double-strand breaks initiate recombination 424 15.5 Recombining chromosomes are connected by the synaptonemal complex 425 XIV CONTENTS By Book_Crazy [IND]
  10. 10. 15.6 The synaptonemal complex forms after double-strand breaks 426 15.7 Pairing and synaptonemal complex formation are independent 428 15.8 The bacterial RecBCD system is stimulated by chi sequences 429 15.9 Strand-transfer proteins catalyze single-strand assimilation . 431 15.10 The Ruv system resolves Holliday junctions 433 15.1 1 Gene conversion accounts for interallelic recombination 434 15.12 Supercoiling affects the structure of DNA 436 15.13 Topoisomerases relax or introduce supercoils in DNA 438 15.14 Topoisomerases break and reseal strands 440 15.15 Gyrase functions by coil inversion 441 15.16 Specialized recombination involves specific sites 442 15.17 Site-specific recombination involves breakage and reunion 444 15.18 Site-specific recombination resembles topoisomerase activity 445 15.19 Lambda recombination occurs in an intasome 446 15.20 Repair systems correct damage to DNA 447 15.21 Excision repair systems in E. coli 450 15.22 Base flipping is used by methylases and glycosylases 451 15.23 Error-prone repair and mutator phenotypes 452 15.24 Controlling the direction of mismatch repair 453 15.25 Recombination-repair systems in E. coli 455 15.26 Recombination is an important mechanism to recover froTn replication errors 456 15.27 RecA triggers the SOS system 457 15.28 Eukaryotic cells have conserved repair systems 459 15.29 A common system repairs double-strand breaks 460 15.30 Summary 462 16 Transposons 16.1 Introduction 467 16.2 Insertion sequences are simple transposition modules 468 16.3 Composite transposons have IS modules 470 16.4 Transposition occurs by both replicative and nonreplicative mechanisms 471 16.5 Transposons cause rearrangement of DNA 473 16.6 Common intermediates for transposition 474 16.7 Replicative transposition proceeds through a cointegrate 475 16.8 Nonreplicative transposition proceeds by breakage and reunion 476 16.9 TnA transposition requires transposase and resolvase 478 16.10 Transposition of Tn10 has multiple controls 480 16.11 Controlling elements in maize cause breakage and rearrangements 482 16.12 Controlling elements form families of transposons 483 16.13 Spm elements influence gene expression 486 16.14 The role of transposable elements in hybrid dysgenesis 487 16.15 P elements are activated in the germline 488 16.16 Summary 490 CONTENTS XV 17 Retroviruses and retroposons 17.1 Introduction 493 17.2 The retrovirus life cycle involves transposition-like events 493 17.3 Retroviral genes code for polyproteins 494 17.4 Viral DNA is generated by reverse transcription 496 17.5 Viral DNA integrates into the chromosome 498 17.6 Retroviruses may transduce cellular sequences 499 17.7 Yeast Ty elements resemble retroviruses 500 17.8 Many transposable elements reside in D. melanogaster 502 17.9 Retroposons fall into three classes 504 17.10 The Alu family has many widely dispersed members 506 17.11 Processed pseudogenes originated as substrates for transposition 507 17.12 LINES use an endonuclease to generate a priming end 508 17.13 Summary 509 By Book_Crazy [IND]
  11. 11. 18 Rearrangement of DNA 18.1 Introduction 513 18.2 The mating pathway is triggered by pheromone-receptor interactions 514 18.3 The mating response activates a G protein 515 18.4 The signal is passed to a kinase cascade 516 18.5 Yeast can switch silent and active loci for mating type 517 18.6 The MAT locus codes for regulator proteins 519 18.7 Silent cassettes at HML and HMR are repressed 521 18.8 Unidirectional transposition is initiated by the recipient MAT locus 522 18.9 Regulation of HO expression controls switching 523 18.10 Trypanosomes switch the VSG frequently during infection 525 18.11 New VSG sequences are generated by gene switching 526 18.12 VSG genes have an unusual structure 528 18.13 The bacterial Ti plasmid causes crown gall disease in plants 529 18.14 T-DNA carries genes required for infection 530 18.15 Transfer of T-DNA resembles bacterial conjugation 532 18.16 DNA amplification generates extra gene copies 534 18.17 Transfection introduces exogenous DNA into cells 537 18.18 Genes can be injected into animal eggs 538 18.19 ES cells can be incorporated into embryonic mice - 540 18.20 Gene targeting allows genes to be replaced or knocked out 541 18.21 Summary 542 Part 5 The Nucleus 19 Chromosomes 19.1 Introduction 545 19.2 Viral genomes are packaged into their coats 5 4 6 19.3 The bacterial genome is a nucleoid 549 19.4 The bacterial genome is supercoiled 550 19.5 Eukaryotic DNA has loops and domains attached to a scaffold 551 19.6 Specific sequences attach DNA to an interphase matrix 552 19.7 Chromatin is divided into euchromatin and heterochromatin 553 19.8 Chromosomes have banding patterns 555 19.9 Lampbrush chromosomes are extended 556 19.10 Polytene chromosomes form bands 557 19.11 Polytene chromosomes expand at sites of gene expression 558 19.12 The eukaryotic chromosome is a segregation device 559 19.13 Centromeres have short DNA sequences in S. cerevisiae 560 19.14 The centromere binds a protein complex 561 19.15 Centromeres may contain repetitious DNA 562 A^."§ Telomeres have simple repeating sequences 563 19.17 Telomeres seal the chromosome ends 564 19.18 Telomeres are synthesized by a ribonucleoprotein enzyme 565 19.19 Telomeres are essential for survival 566 19.20 Summary 567 20 Nucleosomes 20.1 Introduction 571 20.2 The nucleosome is the subunit of all chromatin 572 20.3 DNA is coiled in arrays of nucleosomes 573 20.4 Nucleosomes have a common structure 574 20.5 DNA structure varies on the nucleosomal surface 576 20.6 The periodicity of DNA changes on the nucleosome 577 20.7 The path of nucleosomes in the chromatin fiber 578 20.8 Organization of the histone octamer 579 20.9 The N-terminat tails of histories are modified 581 20.10 Reproduction of chromatin requires assembly of nucleosomes 582 20.11 Do nucleosomes lie at specific positions? 585 XVI CONTENTS By Book_Crazy [IND]
  12. 12. 20.12 Are transcribed genes organized in nucleosomes? 587 20.13 Histone octamers are displaced by transcription 588 20.14 DNAase hypersensitive sites change chromatin structure 590 20.15 Domains define regions that contain active genes 592 20.16 An LCR may control a domain 593 20.17 Summary 594 21 Promoters and enhancers 21.1 Introduction 597 21.2 Eukaryotic RNA polymerases consist of many subunits 599 21.3 Promoter elements are defined by mutations and footprinting 600 21.4 RNA polymerase I has a bipartite promoter 601 21.5 RNA polymerase III uses both downstream and upstream promoters 602 21.6 TF|||B is the commitment factor for pol III promoters 603 21.7 The startpoint for RNA polymerase II' 605 21.8 TBP is a universal factor 606 21.9 TBP binds DNA in an unusual way 607 21.10 The basal apparatus assembles at the promoter 608 21.11 Initiation is followed by promoter clearance 610 21.12 A connection between transcription and repair _ 611 21.13 Short sequence elements bind activators 613 21.14 Promoter construction is flexible but context can be important 614 21.15 Enhancers contain bidirectional elements that assist initiation 615 21.16 Enhancers contain the same elements that are found at promoters 61 6 21.17 Enhancers work by increasing the concentration of activators near the promoter 617 21.18 Gene expression is associated with demethylation 618 21.19 CpG islands are regulatory targets 620 21.20 Insulators block the actions of enhancers and heterochromatin 621 21.21 Insulators can define a domain 622 21.22 Insulators may act in one direction 623 21.23 Insulators can vary in strength 624 21.24 What constitutes a regulatory domain? 625 21.25 Summary 626 22 Activating transcription 22.1 Introduction 631 22.2 There are several types of transcription factors 632 22.3 Independent domains bind DNA and activate transcription 633 22.4 The two hybrid assay detects protein-protein interactions 635 22.5 Activators interact with the basal apparatus 636 22.6 Some promoter-binding proteins are repressors 638 22.7 Response elements are recognized by activators 639 22.8 There are many types of DNA-binding domains 641 22.9 A zinc finger motif is a DNA-binding domain 642 22.10 Steroid receptors are activators 643 22.1 1 Steroid receptors have zinc fingers 644 22.12 Binding to the response element is activated by ligand-binding 645 22.13 Steroid receptors recognize response elements by a combinatorial code 646 22.14 Homeodomains bind related targets in DNA 647 22.15 Helix-loop-helix proteins interact by combinatorial association 649 22.16 Leucine zippers are involved in dimer formation 651 22.17 Summary 652 23 Controlling chromatin structure 23.1 Introduction 657 23.2 Chromatin can have alternative states 658 23.3 Chromatin remodeling is an active process 659 23.4 Nucleosome organization may be changed at the promoter 661 23.5 Histone modification is a key event 662 23.6 Histone acetylation occurs in two circumstances 663 23.7 Acetylases are associated with activators 665 CONTENTS XVII By Book_Crazy [IND]
  13. 13. 23.8 Deacetylases are associated with repressors 666 23.9 Methylation of histones and DNA is connected 667 23.10 Chromatin states are interconverted by modification 668 23.11 Promoter activation involves an ordered series of events 668 23.12 Histone phosphorylation affects chromatin structure 669 23.13 Heterochromatin propagates from a nucleation event 670 23.14 Some common motifs are found in proteins that modify chromatin 671 23.15 Heterochromatin depends on interactions with histones 672 23.16 Polycomb and trithorax are antagonistic repressors and activators 674 23.17 X chromosomes undergo global changes 676 23.18 Chromosome condensation is caused by condensins 678 23.19 DNA methylation is perpetuated by a maintenance methylase 680 23.20 DNA methylation is responsible for imprinting 681 23.21 Oppositely imprinted genes can be controlled by a single center 683 23.22 Epigenetic effects can be inherited 683 23.23 Yeast prions show unusual inheritance 685 23.24 Prions cause diseases in mammals 687 23.25 Summary 689 24 RNA splicing and processing 24.1 Introduction 697 24.2 Nuclear splice junctions are short sequences 698 24.3 Splice junctions are read in pairs 699 24.4 pre-mRNA splicing proceeds through a lariat 701 24.5 snRNAs are required for splicing 702 24.6 U1 snRNP initiates splicing 704 24.7 The E complex can be formed by intron definition or exon definition 706 24.8 5 snRNPs form the spliceosome 707 24.9 An alternative splicing apparatus uses different snRNPs 709 24.10 Splicing is connected to export of mRNA 709 24.11 Group il introns autosplice via lariat formation 710 24.12 Alternative splicing involves differential use of splice junctions 712 24.13 frans-splicing reactions use small RNAs 714 24.14 Yeast tRNA splicing involves cutting and rejoining 716 24.15 The splicing endonuclease recognizes tRNA 717 24.16 tRNA cleavage and ligation are separate reactions 718 24.17 The unfolded protein response is related to tRNA splicing 719 24.18 The 3' ends of poll and poll 11 transcripts are generated by termination 720 24.19 The 3' ends of mRNAs are generated by cleavage and polyadenylation 721 24.20 Cleavage of the 3' end of histone mRNA may require a small RNA 723 24.21 Production of rRNA requires cleavage events 723 24.22 Small RNAs are required for rRNA processing 724 24.23 Summary 725 25 Catalytic RNA 25.1 Introduction 731 25.2 Group I introns undertake self-splicing by transesterification 732 25.3 Group I introns form a characteristic secondary structure 734 25.4 Ribozymes have various catalytic activities 735 25.5 Some group I introns code for endonucleases that sponsor mobility 737 25.6 Some group II introns code for reverse transcriptases 739 25.7 The catalytic activity of RNAase P is due to RNA 740 25.8 Viroids have catalytic activity 740 25.9 RNA editing occurs at individual bases 742 25.10 RNA editing can be directed by guide RNAs 743 25.11 Protein splicing is autocatalytic 746 25.12 Summary 747 26 Immune diversity 26.1 Introduction 751 26.2 Clonal selection amplifies lymphocytes that respond to individual antigens 753 XVIII CONTENTS By Book_Crazy [IND]
  14. 14. 26.3 Immunoglobulin genes are assembled from their parts in lymphocytes 754 26.4 Light chains are assembled by a single recombination 757 26.5 Heavy chains are assembled by two recombinations 758 26.6 Recombination generates extensive diversity 759 26.7 Immune recombination uses two types of consensus sequence 760 26.8 Recombination generates deletions or inversions 761 26.9 The RAG proteins catalyze breakage and reunion 762 26.10 Allelic exclusion is triggered by productive rearrangement 765 26.11 Class switching is caused by DNA recombination 766 26.12 Switching occurs by a novel recombination reaction 768 26.13 Early heavy chain expression can be changed by RNA processing 769 26.14 Somatic mutation generates additional diversity in mouse and man 770 26.15 Somatic mutation is induced by cytidine deaminase and uracil glycosylase 771 26.16 Avian immunoglobulins are assembled from pseudogenes 773 26.17 B cell memory allows a rapid secondary response 774 26.18 T cell receptors are related to immunoglobulins 775 26.19 The T cell receptor functions in conjunction with the MHC 777 26.20 The major histocompatibility locus codes for many genes of the immune system 778 26.21 Innate immunity utilizes conserved signaling pathways 781 26.22 Summary 783 Part 6 Cells 27 Protein trafficking 27.1 Introduction 787 27.2 Oligosaccharides are added to proteins in the ER and Golgi 788 27.3 The Golgi stacks are polarized 790 27.4 Coated vesicles transport both exported and imported proteins 790 27.5 Different types of coated vesicles exist in each pathway 792 27.6 Cisternal progression occurs more slowly than vesicle movement 795 27.7 Vesicles can bud and fuse with membranes 796 27.8 The exocyst tethers vesicles by interacting with a Rab 797 27.9 SNARES are responsible for membrane fusion 798 27.10 The synapse is a model system for exocytosis 800 27.11 Protein localization depends on specific signals 800 27.12 ER proteins are retrieved from the Golgi 802 27.13 Brefeldin A reveals retrograde transport 803 27.14 Vesicles and cargos are sorted for different destinations 804 27.15 Receptors recycle via endocytosis 804 27.16 Internalization signals are short and contain tyrosine 806 27.17 Summary 807 28 Signal transduction 28.1 Introduction 811 28.2 Carriers and channels form water soluble paths through the membrane 813 28.3 Ion channels are selective 814 28.4 Neurotransmitters control channel activity 816 28.5 G proteins may activate or inhibit target proteins 817 28.6 G proteins function by dissociation of the trimer 818 28.7 Protein kinases are important players in signal transduction 819 28.8 Growth factor receptors are protein kinases 821 28.9 Receptors are activated by dimerization 822 28.10 Receptor kinases activate signal transduction pathways 823 28.11 Signaling pathways often involve protein-protein interactions 824 28.12 Phosphotyrosine is the critical feature in binding to an SH2 domain 825 28.13 Prolines are important determinants in recognition sites 826 28.14 The Ras/MAPK pathway is widely conserved 827 28.15 The activation of Ras is controlled by GTP 829 28.16 A MAP kinase pathway is a cascade 830 28.17 What determines specificity in signaling? 832 CONTENTS XIX By Book_Crazy [IND]
  15. 15. 28.18 Activation of a pathway can produce different results 834 28.19 Cyclic AMP and activation of CREB 835 28.20 The JAK-STAT pathway 836 28.21 TGFP signals through Smads 838 28.22 Summary 839 29 Cell cycle and growth regulation 29.1 Introduction 843 29.2 Cycle progression depends on discrete control points 844 29.3 Checkpoints occur throughout the cell cycle 845 29.4 Cell fusion experiments identify cell cycle inducers 846 29.5 M phase kinase regulates entry into mitosis 848 29.6 M phase kinase is a dimer of a catalytic subunit and a regulatory cyclin 849 29.7 Protein phosphorylation and dephosphorylation control the cell cycle 851 29.8 Many cell cycle mutants have been found by screens in yeast 853 29.9 Cdc2 is the key regulator in yeasts 854 29.10 Cdc2 is the only catalytic subunit of the cell cycle activators in S. pombe 855 29.11 CDC28 acts at both START and mitosis in S. cerevisiae 856 29.12 Cdc2 activity is controlled by kinases and phosphatases 858 29.13 DNA damage triggers a checkpoint 861 29.14 The animal cell cycle is controlled by many cdk-cyclin complexes 863 29.15 Dimers are controlled by phosphorylation of cdk subunits and by availability of cyclin subunits 864 29.16 RB is a major substrate for cdk-cyclin complexes 866 29.17 G0/G1 and G1/S transitions involve cdk inhibitors 867 29.18 Protein degradation is important in mitosis 868 29.19 Cohesins hold sister chromatids together 869 29.20 Exit from mitosis is controlled by the location of Cdc14 871 29.21 The cell forms a spindle at mitosis 871 29.22 The spindle is oriented by centrosomes 873 29.23 A monomeric G protein controls spindle assembly 874 29.24 Daughter cells are separated by cytokinesis 875 29.25 Apoptosis is a property of many or all cells 876 29.26 The Fas receptor is a major trigger for apoptosis 876 29.27 A common pathway for apoptosis functions via caspases 878 29.28 Apoptosis involves changes at the mitochondrial envelope 879 29.29 Cytochrome c activates the next stage of apoptosis 880 29.30 There are multiple apoptotic pathways 882 29.31 Summary 882 30 Oncogenes and cancer 30.1 Introduction 889 30.2 Tumor cells are immortalized and transformed 890 30.3 Oncogenes and tumor suppressors have opposite effects 892 30.4 Transforming viruses carry oncogenes 893 30.5 Early genes of DNA transforming viruses have multifunctional oncogenes 893 30.6 Retroviruses activate or incorporate cellular genes 895 30.7 Retroviral oncogenes have cellular counterparts 896 30.8 Quantitative or qualitative changes can explain oncogenicity 898 30.9 Ras oncogenes can be detected in a transfection assay 899 30.10 Ras proto-oncogenes can be activated by mutation at specific positions 900 30.11 Nondefective retroviruses activate proto-oncogenes 901 30.12 Proto-oncogenes can be activated by translocation 902 30.13 The Philadelphia translocation generates a new oncogene 904 30.14 Oncogenes code for components of signal transduction cascades 905 30.15 Growth factor receptor kinases can be mutated to oncogenes 907 30.16 Src is the prototype for the proto-oncogenic cytoplasmic tyrosine kinases 909 30.17 Src activity is controlled by phosphorylation 910 30.18 Oncoproteins may regulate gene expression 912 30.19 RB is a tumor suppressor that controls the cell cycle 915 30.20 Tumor suppressor p53 suppresses growth or triggers apoptosis 917 XX CONTENTS By Book_Crazy [IND]
  16. 16. 30.21 p53 is a DNA-binding protein 919 30.22 p53 is controlled by other tumor suppressors and oncogenes 921 30.23 p53 is activated by modifications of amino acids 922 30.24 Telomere shortening causes cell senescence . 923 30.25 Immortalization depends on loss of p53 925 30.26 Different oncogenes are associated with immortalization and transformation 926 30.27 p53 may affect ageing 929 30.28 Genetic instability is a key event in cancer 930 30.29 Defects in repair systems cause mutations to accumulate in tumors 931 30.30 Summary 932 31 Gradients, cascades, and signaling pathways 31.1 Introduction 939 31.2 Fly development uses a cascade of transcription factors 940 31.3 A gradient must be converted into discrete compartments 941 31.4 Maternal gene products establish gradients in early embryogenesis 943 31.5 Anterior development uses localized gene regulators 945 31.6 Posterior development uses another localized regulator 946 31.7 How are mRNAs and proteins transported and localized? 948 31.8 How are gradients propagated? - 949 31.9 Dorsal-ventral development uses localized receptor-ligand interactions 950 31.10 Ventral development proceeds through Toll 951 31.11 Dorsal protein forms a gradient of nuclear localization 953 31.12 Patterning systems have common features 955 31.13 TGFp/BMPs are diffusible morphogens 956 31.14 Cell fate is determined by compartments that form by the blastoderm stage 957 31.15 Gap genes are controlled by bicoid and by one another 959 31.16 Pair-rule genes are regulated by gap genes 960 31.17 Segment polarity genes are controlled by pair-rule genes 961 31.18 Wingless and engrailed expression alternate in adjacent cells 963 31.19 The wingless/wnt pathway signals to the nucleus 964 31.20 Complex loci are extremely large and involved in regulation 965 31.21 The bithorax complex has frans-acting genes and c/s-acting regulators 968 31.22 The homeobox is a common coding motif in homeotic genes 972 31.23 Summary 975 Glossary 981 Index 1003 CONTENTS XXI By Book_Crazy [IND]
  17. 17. GENES is continuously updated on the web site, www.ergito.com with revisions posted weekly. This allows readers to check for revised sections and relate them to the printed book. The web site can be viewed as either sections from the book or as a slide show of the figures from the book. Some of the figures shown are animated and there are references hyperlinked to the original sources. Other features of the web site include a glossary, sophisticated searches, and ancillary material such as the essays in the Great Experiments and Structures Series. To subscribe to this site, please visit www.ergito.com. By Book_Crazy [IND]
  18. 18. Chapter 1 Genes are DNA 1.1 Introduction The hereditary nature of every living organism is defined by its genome, which consists of a long sequence of nucleic acid that provides the information needed to construct the organism. We use the term "information" because the genome does not itself perform any ac- tive role in building the organism; rather it is the sequence of the indi- vidual subunits (bases) of the nucleic acid that determines hereditary features. By a complex series of interactions, this sequence is used to produce all the proteins of the organism in the appropriate time and place. The proteins either form part of the structure of the organism, or have the capacity to build the structures or to perform the metabolic reactions necessary for life. The genome contains the complete set of hereditary information for any organism. Physically the genome may be divided into a number of different nucleic acid molecules. Functionally it may be divided into genes. Each gene is a sequence within the nucleic acid that represents a single protein. Each of the discrete nucleic acid molecules comprising the genome may contain a large number of genes. Genomes for living organisms may contain as few as <500 genes (for a mycoplasma, a type of bacterium) to as many as >40,000 for Man. In this chapter, we analyze the properties of the gene in terms of its basic molecular construction. Figure 1.1 summarizes the stages in the transition from the historical concept of the gene to the modern defini- tion of the genome. The basic behavior of the gene was defined by Mendel more than a century ago. Summarized in his two laws, the gene was recognized as a "particulate factor" that passes unchanged from parent to progeny. A gene may exist in alternative forms. These forms are called alleles. In diploid organisms, which have two sets of chromosomes, one copy of each chromosome is inherited from each parent. This is the same behavior that is displayed by genes. One of the two copies of each gene is the paternal allele (inherited from the father), the other is the maternal allele (inherited from the mother). The equivalence led to the discovery that chromosomes in fact carry the genes. Introduction SECTION 1.1 1.1 Introduction 1.17 Mutations may cause loss-of-function or gain-of- 1.2 DNA is the genetic material of bacteria function 1.3 DNA is the genetic material of viruses 1.18 A locus may have many different mutant alleles 1.4 DNA is the genetic material of animal cells 1.19 A locus may have more than one wild-type allele 1.5 Polynucleotide chains have nitrogenous bases 1.20 Recombination occurs by physical exchange of linked to a sugar-phosphate backbone DNA 1.6 DNA is a double helix 1.21 The genetic code is triplet 1.7 DNA replication is semiconservative 1.22 Every sequence has three possible reading 1.8 DNA strands separate at the replication fork frames 1.9 Nucleic acids hybridize by base pairing 1.23 Prokaryotic genes are colinear with their proteins 1.10 Mutations change the sequence of DNA 1.24 Several processes are required to express the 1.11 Mutations may affect single base pairs or longer protein product of a gene sequences 1.25 Proteins are frans-acting but sites on DNA are 1.12 The effects of mutations can be reversed c/s-acting 1.13 Mutations are concentrated at hotspots 1.26 Genetic information can be provided by DNA or 1.14 Many hotspots result from modified bases RNA 1.15 A gene codes for a single polypeptide 1.27 Some hereditary agents are extremely small 1.16 Mutations in the same gene cannot complement 1.28 Summary By Book_Crazy [IND]
  19. 19. Each chromosome consists of a linear array of genes. Each gene re- sides at a particular location on the chromosome. This is more formally called a genetic locus. We can then define the alleles of this gene as the different forms that are found at this locus. The key to understanding the organization of genes into chromosomes was the discovery of genetic linkage. This describes the observation that alleles on the same chromosome tend to remain together in the progeny instead of assorting independently as predicted by Mendel's laws. Once the unit of recombination (reassortment) was introduced as the measure of linkage, the construction of genetic maps became possible. On the genetic maps of higher organisms established during the first half of this century, the genes are arranged like beads on a string. They occur in a fixed order, and genetic recombination involves transfer of corresponding portions of the string between homologous chromo- somes. The gene is to all intents and purposes a mysterious object (the bead), whose relationship to its surroundings (the string) is unclear. The resolution of the recombination map of a higher eukaryote is re- stricted by the small number of progeny that can be obtained from each mating. Recombination occurs so infrequently between nearby points that it is rarely observed between different mutations in the same gene. By moving to a microbial system in which a very large number of prog- eny can be obtained from each genetic cross, it became possible to demonstrate that recombination occurs within genes. It follows the same rules that were previously deduced for recombination between genes. Mutations within a gene can be arranged into a linear order, showing that the gene itself has the same linear construction as the array of genes on a chromosome. So the genetic map is linear within as well as between loci: it consists of an unbroken sequence within which the genes reside. This conclusion leads naturally into the modern view that the genetic material of a chromosome consists of an uninterrupted length of DNA representing many genes. A genome consists of the entire set of chromosomes for any particu- lar organism. It therefore comprises a series of DNA molecules (one for each chromosome), each of which contains many genes. The ultimate definition of a genome is to determine the sequence of the DNA of each chromosome. The first definition of the gene as a functional unit followed from the discovery that individual genes are responsible for the production of specific proteins. The difference in chemical nature between the DNA of the gene and its protein product led to the concept that a gene codes for a protein. This in turn led to the discovery of the complex apparatus that allows the DNA sequence of gene to generate the amino acid se- quence of a protein. Understanding the process by which a gene is expressed allows us to make a more rigorous definition of its nature. Figure 1.2 shows the basic theme of this book. A gene is a sequence of DNA that produces an- other nucleic acid, RNA. The DNA has two strands of nucleic acid, and the RNA has only one strand. The sequence of the RNA is determined by the sequence of the DNA (in fact, it is identical to one of the DNA strands). In many, but not in all cases, the RNA is in turn used to direct production of a protein. Thus a gene is a sequence of DNA that codes for an RNA; in protein-coding genes, the RNA in turn codes for a protein. From the demonstration that a gene consists of DNA, and that a chromosome consists of a long stretch of DNA representing many genes, we move to the overall organization of the genome in terms of its DNA sequence. In 2 The interrupted gene we take up in more detail the organization of the gene and its representation in proteins. In 3 The content of the genome we consider the total number of genes, and in 4 Clusters and repeats we discuss other components of the genome and the maintenance of its organization. CHAPTER 1 Genes are DNA By Book_Crazy [IND]
  20. 20. 1.2 DNA is the genetic material of bacteria The idea that genetic material is nucleic acid had its roots in the discovery of transformation in 1928. The bacterium Pneumococ- cus kills mice by causing pneumonia. The virulence of the bacterium is determined by its capsular polysaccharide. This is a component of the surface that allows the bacterium to escape destruction by the host. Sev- eral types (I, II, III) of Pneumococcus have different capsular polysaccharides. They have a smooth (S) appearance. Each of the smooth Pneumococcal types can give rise to variants that fail to produce the capsular polysaccharide. These bacteria have a rough (R) surface (consisting of the material that was beneath the capsular polysaccharide). They are aviru- lent. They do not kill the mice, because the absence of the poly- saccharide allows the animal to destroy the bacteria. When smooth bacteria are killed by heat treatment, they lose their ability to harm the animal. But inactive heat-killed S bac- teria and the ineffectual variant R bacteria together have a quite different effect from either bacterium by itself. Figure 1.3 shows that when they are jointly injected into an animal, the mouse dies as the result of a Pneumococcal infection. Virulent S bacteria can be recovered from the mouse postmortem. In this experiment, the dead S bacteria were of type III. The live R bacteria had been derived from type II. The virulent bacteria recovered from the mixed infection had the smooth coat of type III. So some prop- erty of the dead type III S bacteria can transform the live R bacteria so that they make the type III capsular polysaccharide, and as a result be- come virulent. Figure 1.4 shows the identification of the component of the dead bacteria responsible for transformation. This was called the transform- ing principle. It was purified by developing a cell-free system, in which extracts of the dead S bacteria could be added to the live R bacteria be- fore injection into the animal. Purification of the transforming principle in 1944 showed that it is deoxyribonucleic acid (DNA). 1.3 DNA is the genetic material of viruses Having shown that DNA is the genetic material of bacteria, the next step was to demonstrate that DNA provides the genetic ma- terial in a quite different system. Phage T2 is a virus that infects the DNA is the genetic material of bacteria SECTION 1.2 i Figure 1.3 Neither heat-killed S-type nor : live R-type bacteria can kill mice, but : simultaneous infection of them both can : kill mice just as effectively as the live : S-type. i Key Concepts ; ; * Phage infection proved that DNA is the genetic material of : • viruses. When the DNA and protein components of bacteriophages i ; are labeled with different radioactive isotopes, only the DNA is : transmitted to the progeny phages produced by infecting bacteria. • I Key Concepts j I * Bacterial transformation provided the first proof that DNA is the : : genetic material. Genetic properties can be transferred from one • : bacterial strain to another by extracting DNA from the first strain : : and adding it to the second strain. • By Book_Crazy [IND]
  21. 21. I bacterium E. coli. When phage particles are added to bacteria, they ad- sorb to the outside surface, some material enters the bacterium, and then -20 minutes later each bacterium bursts open (lyses) to release a large number of progeny phage. Figure 1.5 illustrates the results of an experiment in 1952 in which bacteria were infected with T2 phages that had been radioactively la- beled either in their DNA component (with 32 P) or in their protein com- ponent (with 35 S). The infected bacteria were agitated in a blender, and two fractions were separated by centrifugation. One contained the empty phage coats that were released from the surface of the bacteria. The other fraction consisted of the infected bacteria themselves. Most of the 32 P label was present in the infected bacteria. The progeny phage particles produced by the infection contained ~30% of the original 32 P label. The progeny received very little—less than 1%—of the protein contained in the original phage population. The phage coats consist of protein and therefore carried the 35 S radioac- tive label. This experiment therefore showed directly that only the DNA of the parent phages enters the bacteria and then becomes part of the progeny phages, exactly the pattern of inheritance expected of genetic material. A phage (virus) reproduces by commandeering the machinery of an infected host cell to manufacture more copies of itself. The phage pos- sesses genetic material whose behavior is analogous to that of cellular genomes: its traits are faithfully reproduced, and they are subject to the «w«M«,-re«l«&,tlM»i fjeaw-isij. isaheavitaiJGe,-Xbe, case, of Ti E£mfblC6S- the- 2£n- eral conclusion that the genetic material is BNA, wriemeir part of me genome of a cell or virus. 1.4 DNA is the genetic material of animal cells When DNA is added to populations of single eukaryotic cells growing in culture, the nucleic acid enters the cells, and in some of them results in the production of new proteins. When a purified DNA is used, its incorporation leads to the production of a particular protein. Figure 1.6 depicts one of the standard systems. Although for historical reasons these experiments are described as transfection when performed with eukaryotic cells, they are a direct counterpart to bacterial transformation. The DNA that is introduced into the recipient cell becomes part of its genetic material, and is inher- ited in the same way as any other part. Its expression confers a new trait upon the cells (synthesis of thymidine kinase in the example of the fig- ure). At first, these experiments were successful only with individual cells adapted to grow in a culture medium. Since then, however, DNA has been introduced into mouse eggs by microinjection; and it may be- come a stable part of the genetic material of the mouse (see 18.18 Genes can be injected into animal eggs). Such experiments show directly not only that DNA is the genetic material in eukaryotes, but also that it can be transferred between dif- ferent species and yet remain functional. The genetic material of all known organisms and many viruses is DNA. However, some viruses use an alternative type of nucleic acid, CHAPTER 1 Genes are DNA By Book_Crazy [IND]
  22. 22. ribonucleic acid (RNA), as the genetic material. The general principle of the nature of the genetic material, then, is that it is always nucleic acid; in fact, it is DNA except in the RNA viruses. 1.5 Polynucleotide chains have nitrogenous bases linked to a sugar-phosphate backbone The basic building block of nucleic acids is the nucleotide. This has three components: • a nitrogenous base; • a sugar; • and a phosphate. The nitrogenous base is a purine or pyrimidine ring. The base is linked to position 1 on a pentose sugar by a glycosidic bond from Ni of pyrimidines or N9 of purines. To avoid ambiguity between the number- ing systems of the heterocyclic rings and the sugar, positions on the pentose are given a prime (')• Nucleic acids are named for the type of sugar; DNA has 2'-deoxyri- bose, whereas RNA has ribose. The difference is that the sugar in RNA has an OH group at the 2' position of the pentose ring. The sugar can be linked by its 5' or 3' position to a phosphate group. A nucleic acid consists of a long chain of nucleotides. Figure 1.7 shows that the backbone of the polynucleotide chain consists of an al- ternating series of pentose (sugar) and phosphate residues. This is con- structed by linking the 5' position of one pentose ring to the 3' position of the next pentose ring via a phosphate group. So the sugar-phosphate backbone is said to consist of 5'-3' phosphodiester linkages. The ni- trogenous bases "stick out" from the backbone. Each nucleic acid contains 4 types of base. The same two purines, adenine and guanine, are present in both DNA and RNA. The two pyrim- idines in DNA are cytosine and thymine; in RNA uracil is found instead of thymine. The only difference between uracil and thymine is the pres- ence of a methyl substituent at position C5. The bases are usually referred to by their initial letters. DNA contains A, G, C, T, while RNA contains A, G, C, U. The terminal nucleotide at one end of the chain has a free 5' group; the terminal nucleotide at the other end has a free 3' group. It is con- ventional to write nucleic acid sequences in the 5'—>3' direction—that is, from the 5' terminus at the left to the 3' terminus at the right. Polynucleotide chains have nitrogenous bases linked to a sugar-phosphate backbone SECTION 1.5 By Book_Crazy [IND]
  23. 23. 1.6 DNA is a double helix Figure 1.8 The double helix maintains a constant width because purines always face pyrimidines in the complementary A-T and G-C base pairs. The sequence in the figure is T-A, C-G, A-T, G-C. L The observation that the bases are present in different amounts in the DNAs of different species led to the concept that the sequence of bases is the form in which genetic information is carried. By the 1950s, the concept of genetic information was common: the twin prob- lems it posed were working out the structure of the nucleic acid, and ex- plaining how a sequence of bases in DNA could represent the sequence of amino acids in a protein. Three notions converged in the construction of the double helix model for DNA by Watson and Crick in 1953: • X-ray diffraction data showed that DNA has the form of a regular helix, making a complete turn every 34 A (3.4 nm), with a diameter of ~20 A (2 nm). Since the distance between adjacent nucleotides is 3.4 A, there must be 10 nucleotides per turn. • The density of DNA suggests that the helix must contain two polynucleotide chains. The constant diameter of the helix can be explained if the bases in each chain face inward and are restricted so that a purine is always opposite a pyrimidine, avoiding partner- ships of purine-purine (too wide) or pyrimidine-pyrimidine (too narrow). • Irrespective of the absolute amounts of each base, the propor- tion of G is always the same as the proportion of C in DNA, and the proportion of A is always the same as that of T. So the composition of any DNA can be described by the proportion of its bases that is G + C. This ranges from 26% to 74% for different species. Watson and Crick proposed that the two polynucleotide chains in the double helix associate by hydrogen bonding be- tween the nitrogenous bases. G can hydrogen bond specifically only with C, while A can bond specifically only with T. These reactions are described as base pairing, and the paired bases (G with C, or A with T) are said to be complementary. The model proposed that the two polynucleotide chains run in opposite directions (antiparallel), as illustrated in Figure 1.8. Looking along the helix, one strand runs in the 5'—>3' direc- tion, while its partner runs 3'—»5'. The sugar-phosphate backbone is on the outside and carries negative charges on the phosphate groups. When DNA is in so- lution in vitro, the charges are neutralized by the binding of metal ions, typically by Na+ . In the cell, positively charged pro- teins provide some of the neutralizing force. These proteins play an im- portant role in determining the organization of DNA in the cell. The bases lie on the inside. They are flat structures, lying in pairs perpendicular to the axis of the helix. Consider the double helix in CHAPTER 1 Genes are DNA By Book_Crazy [IND]
  24. 24. terms of a spiral staircase: the base pairs form the treads, as illustrated schematically in Figure 1.9. Proceeding along the helix, bases are stacked above one another, in a sense like a pile of plates. Each base pair is rotated ~36° around the axis of the helix relative to the next base pair. So ~10 base pairs make a complete turn of 360°. The twisting of the two strands around one another forms a double helix with a minor groove (~12 A across) and a major groove (~22 A across), as can be seen from the scale model of Figure 1.10. The double helix is right-handed; the turns run clockwise looking along the helical axis. These features represent the accepted model for what is known as the B-formofDNA. It is important to realize that the B-form represents an average, not a precisely specified structure. DNA structure can change locally. If it has more base pairs per turn it is said to be overwound; if it has fewer base pairs per turn it is underwound. Local winding can be affected by the overall conformation of the DNA double helix in space or by the binding of proteins to specific sites. 1.7 DNA replication is semiconservative It is crucial that the genetic material is reproduced accurately. Be- cause the two polynucleotide strands are joined only by hydrogen bonds, they are able to separate without requiring breakage of covalent bonds. The specificity of base pairing suggests that each of the sepa- rated parental strands could act as a template strand for the synthesis of a complementary daughter strand. Figure 1.11 shows the principle that a new daughter strand is assembled on each parental strand. The sequence of the daughter strand is dictated by the parental strand; an A in the parental strand causes a T to be placed in the daughter strand, a parental G directs incorporation of a daughter C, and so on. The top part of the figure shows a parental (unreplicated) duplex that consists of the original two parental strands. The lower part shows the two daughter duplexes that are being produced by complementary base pairing. Each of the daughter duplexes is identical in sequence with the original parent, and contains one parental strand and one newly synthe- sized strand. The structure of DNA carries the information needed to perpetuate its sequence. The consequences of this mode of replication are illustrated in Figure 1.12. The parental duplex is replicated to form two daughter duplexes, each of which consists of one parental strand and one (newly synthesized) daughter strand. The unit conserved from one generation to the next is one of the two individual strands com- prising the parental duplex. This behavior is called semiconservative replication. The figure illustrates a prediction of this model. If the parental DNA "heavy,, density label because the organism has been grown in £WA , r stJinfconservatfve I SECTION' T.7 By Book_Crazy [IND]
  25. 25. medium containing a suitable isotope (such as 15 N), its strands can be distinguished from those that are synthesized when the organism is transferred to a medium containing normal "light" isotopes. The parental DNA consists of a duplex of two heavy strands (red). After one generation of growth in light medium, the duplex DNA is "hybrid" in density—it consists of one heavy parental strand (red) and one light daughter strand (blue). After a second generation, the two strands of each hybrid duplex have separated; each gains a light part- ner, so that now half of the duplex DNA remains hybrid while half is entirely light (both strands are blue). The individual strands of these duplexes are entirely heavy or en- tirely light. This pattern was confirmed experimentally in the Meselson- Stahl experiment of 1958, which followed the semiconservative replication of DNA through three generations of growth of E. coll. When DNA was. extracted from bacteria and its density measured by centrifugation, the DNA formed bands corresponding to its density— heavy for parental, hybrid for the first generation, and half hybrid and half light in the second generation. 1.8 DNA strands separate at the replication fork Key Concepts • Replication of DNA is undertaken by a complex of enzymes that separate the parental strands and synthesize the daughter strands. • The replication fork is the point at which the parental strands are separated. • The enzymes that synthesize DNA are called DNA polymerases; the enzymes that synthesize RNA are RNA polymerases. • Nucleases are enzymes that degrade nucleic acids; they include DNAases and RNAases, and can be divided into endonucleases and exonucleases. Replication requires the two strands of the parental duplex to sepa- rate. However, the disruption of structure is only transient and is reversed as the daughter duplex is formed. Only a small stretch of the duplex DNA is separated into single strands at any moment. The helical structure of a molecule of DNA engaged in replication is illustrated in Figure 1.13. The nonreplicated region consists of the parental duplex, opening into the replicated region where the two daughter duplexes have formed. The double helical structure is dis- rupted at the junction between the two regions, which is called the replication fork. Replication involves movement of the replication fork along the parental DNA, so there is a continuous unwinding of the parental strands and rewinding into daughter duplexes. The synthesis of nucleic acids is catalyzed by specific enzymes, which recognize the template and undertake the task of catalyzing the addition of subunits to the polynucleotide chain that is being synthe- sized. The enzymes are named according to the type of chain that is syn- thesized: DNA polymerases synthesize DNA, and RNA polymerases synthesize RNA. Degradation of nucleic acids also requires specific enzymes: deoxyribonucleases (DNAases) degrade DNA, and ribonucleases (RNAases) degrade RNA. The nucleases fall into the general classes of exonucleases and endonucleases: 8 CHAPTER 1 Genes are DNA By Book_Crazy [IND]
  26. 26. Endonucleases cut individual bonds within RNA or DNA molecules, generating discrete fragments. Some DNAases cleave both strands of a duplex DNA at the target site, while others cleave only one of the two strands. Endonucleases are involved in cutting reactions, as shown in Figure 1.14. Exonucleases remove residues one at a time from the end of the mol- ecule, generating mononucleotides. They always function on a single nucleic acid strand, and each exonuclease proceeds in a specific di- rection, that is, starting at either a 5' or at a 3' end and proceeding to- ward the other end. They are involved in trimming reactions, as shown in Figure 1.15. 1.9 Nucleic acids hybridize by base pairing Key Concepts • Heating causes the two strands of a DNA duplex to separate. • The Tm is the midpoint of the temperature range for denaturation. • Complementary single strands can renature when the temperature is reduced. • Denaturation and renaturation/hybridization can occur with DNA-DNA, DNA-RNA, or RNA-RNA combinations, and can be intermolecular or intramolecular. • The ability of two single-stranded nucleic acid preparations to hybridize is a measure of their complementarity. Acrucial property of the double helix is the ability to separate the two strands without disrupting covalent bonds. This makes it pos- sible for the strands to separate and reform under physiological condi- tions at the (very rapid) rates needed to sustain genetic functions. The specificity of the process is determined by complementary base pairing. The concept of base pairing is central to all processes involving nu- cleic acids. Disruption of the base pairs is a crucial aspect of the func- tion of a double-stranded molecule, while the ability to form base pairs is essential for the activity of a single-stranded nucleic acid. Figure 1.16 shows that base pairing enables complementary single-stranded nucleic acids to form a duplex structure. • An intramolecular duplex region can form by base pairing between two complementary sequences that are part of a single-stranded molecule. • A single-stranded molecule may base pair with an independent, com- plementary single-stranded molecule to form an intermolecular duplex. Formation of duplex regions from single-stranded nucleic acids is most important for RNA, but single-stranded DNA also exists (in the form of viral genomes). Base pairing between independent complemen- tary single strands is not restricted to DNA-DNA or RNA-RNA, but can also occur between a DNA molecule and an RNA molecule. The lack of covalent links between complementary strands makes it possible to manipulate DNA in vitro. The noncovalent forces that stabi- lize the double helix are disrupted by heating or by exposure to low salt concentration. The two strands of a double helix separate entirely when all the hydrogen bonds between them are broken. The process of strand separation is called denaturation or (more colloquially) melting. ("Denaturation" is also used to describe loss of Nucleic acids hybridize by base pairing SECTION 1.9 By Book_Crazy [IND]
  27. 27. authentic protein structure; it is a general term implying that the natural conformation of a macromolecule has been converted to some other form.) Denaturation of DNA occurs over a narrow temperature range and results in striking changes in many of its physical properties. The mid- point of the temperature range over which the strands of DNA separate is called the melting temperature (Tm). It depends on the proportion of GC base pairs. Because each G-C base pair has three hydrogen bonds, it is more stable than an A-T base pair, which has only two hydrogen bonds. The more G-C base pairs are contained in a DNA, the greater the energy that is needed to separate the two strands. In solution under physiological conditions, a DNA that is 40% G-C—a value typical of mammalian genomes—denatures with a Tm of about 87°C. So duplex DNA is stable at the temperature prevailing in the cell. The denaturation of DNA is reversible under appropriate conditions. The ability of the two separated complementary strands to reform into a double helix is called renaturation. Renaturation depends on specific base pairing between the complementary strands. Figure 1.17 shows that the reaction takes place in two stages. First, single strands of DNA in the solution encounter one another by chance; if their sequences are complementary, the two strands base pair to generate a short double- helical region. Then the region of base pairing extends along the mole- cule by a zipper-like effect to form a lengthy duplex molecule. Renaturation of the double helix restores the original properties that were lost when the DNA was denatured. Renaturation describes the reaction between two complementary se- quences that were separated by denaturation. However, the technique can be extended to allow any two complementary nucleic acid se- quences to react with each other to form a duplex structure. This is sometimes called annealing, but the reaction is more generally de- scribed as hybridization whenever nucleic acids of different sources are involved, as in the case when one preparation consists of DNA and the other consists of RNA. The ability of two nucleic acid preparations to hybridize constitutes a precise test for their complementarity since only complementary sequences can form a duplex structure. The principle of the hybridization reaction is to expose two single- stranded nucleic acid preparations to each other and then to measure the amount of double-stranded material that forms. Figure 1.18 illustrates a procedure in which a DNA preparation is denatured and the single strands are adsorbed to a filter. Then a second denatured DNA (or RNA) preparation is added. The filter is treated so that the second preparation can adsorb to it only if it is able to base pair with the DNA that was originally adsorbed. Usually the second preparation is radioac- tively labeled, so that the reaction can be measured as the amount of ra- dioactive label retained by the filter. The extent of hybridization between two single-stranded nucleic acids is determined by their complementarity. Two sequences need not be perfectly complementary to hybridize. If they are closely related but not identical, an imperfect duplex is formed in which base pairing is in- terrupted at positions where the two single strands do not correspond. 1.10 Mutations change the sequence of DNA Key Concepts * All mutations consist of changes in the sequence of DNA. • Mutations may occur spontaneously or may be induced by mutagens. 10 CHAPTER 1 Genes are DNA By Book_Crazy [IND]
  28. 28. Mutations provide decisive evidence that DNA is the genetic ma- terial. When a change in the sequence of DNA causes an alter- ation in the sequence of a protein, we may conclude that the DNA codes for that protein. Furthermore, a change in the phenotype of the organ- ism may allow us to identify the function of the protein. The existence of many mutations in a gene may allow many variant forms of a protein to be compared, and a detailed analysis can be used to identify regions of the protein responsible for individual enzymatic or other functions. All organisms suffer a certain number of mutations as the result of normal cellular operations or random interactions with the environ- ment. These are called spontaneous mutations; the rate at which they occur is characteristic for any particular organism and is sometimes called the background level. Mutations are rare events, and of course those that damage a gene are selected against during evolution. It is therefore difficult to obtain large numbers of spontaneous mutants to study from natural populations. The occurrence of mutations can be increased by treatment with cer- tain compounds. These are called mutagens, and the changes they cause are referred to as induced mutations. Most mutagens act directly by virtue of an ability either to modify a particular base of DNA or to become in- corporated into the nucleic acid. The effectiveness of a mutagen is judged by how much it increases the rate of mutation above background. By using mutagens, it becomes possible to induce many changes in any gene. Spontaneous mutations that inactivate gene function occur in bacterio- phages and bacteria at a relatively constant rate of 3-4 x 1(T3 per genome per generation. Given the large variation in genome sizes between bacte- riophages and bacteria, this corresponds to wide differences in the muta- tion rate per base pair. This suggests that the overall rate of mutation has been subject to selective forces that have balanced the deleterious effects of most mutations against the advantageous effects of some mutations. This conclusion is strengthened by the observation that an archaeal mi- crobe that lives under harsh conditions of high temperature and acidity (which are expected to damage DNA) does not show an elevated mutation rate, but in fact has an overall mutation rate just below the average range. Figure 1.19 shows that in bacteria, the mutation rate corresponds to ~1(T6 events per locus per generation or to an average rate of change per base pair of 10~9 -10~10 per generation. The rate at individual base pairs varies very widely, over a 10,000 fold range. We have no accurate measurement of the rate of mutation in eukaryotes, although usually it is thought to be somewhat similar to that of bacteria on a per-locus per- generation basis. We do not know what proportion of the spontaneous events results from point mutations. 1.11 Mutations may affect single base pairs or longer sequences Key Concepts • A point mutation changes a single base pair. • Point mutations can be caused by the chemical conversion of one base into another or by mistakes that occur during replication. • A transition replaces a G-C base pair with an A-T base pair or vice-versa. • A transversion replaces a purine with a pyrimidine, such as changing A-T to T-A. • Insertions are the most common type of mutation, and result from the movement of transposable elements. Mutations may affect single base pairs or longer sequences SECTION 1.11 11 By Book_Crazy [IND]
  29. 29. • Chemical modification of DNA directly changes one base into a dif- ferent base. • A malfunction during the replication of DNA causes the wrong base to be inserted into a polynucleotide chain during DNA synthesis. Point mutations can be divided into two types, depending on the nature of the change when one base is substituted for another: • The most common class is the transition, comprising the substitution of one pyrimidine by the other, or of one purine by the other. This re- places a GC pai* with an AT pair or vice versa. • The less common class is the transversion, in which a purine is re- placed by a pyrimidine or vice versa, so that an AT pair becomes a T A or C G pair. The effects of nitrous acid provide a classic example of a transition caused by the chemical conversion of one base into another. Figure 1.20 shows that nitrous acid performs an oxidative deamination that converts cytosine into uracil. In the replication cycle following the tran- sition, the U pairs with an A, instead of with the G with which the orig- inal C would have paired. So the CG pair is replaced by a TA pair when the A pairs with the T in the next replication cycle. (Nitrous acid also deaminates adenine, causing the reverse transition from AT to GC.) Transitions are also caused by base mispairing, when unusual part- ners pair in defiance of the usual restriction to Watson-Crick pairs. Base mispairing usually occurs as an aberration resulting from the in- corporation into DNA of an abnormal base that has ambiguous pairing properties. Figure 1.21 shows the example of bromouracil (BrdU), an analog of thymine that contains a bromine atom in place of the methyl group of thymine. BrdU is incorporated into DNA in place of thymine. But it has ambiguous pairing properties, because the presence of the bromine atom allows a shift to occur in which the base changes struc- ture from a keto (=O) form to an enol (-OH) form. The enol form can base pair with guanine, which leads to substitution of the original AT pair by a GC pair. The mistaken pairing can occur either during the original incor- poration of the base or in a subsequent replication cycle. The transition is induced with a certain probability in each replication cycle, so the incorporation of BrdU has continuing effects on the sequence of DNA. Point mutations were thought for a long time to be the principal means of change in individual genes. However, we now know that in- sertions of stretches of additional material are quite frequent. The source of the inserted material lies with transposable elements, se- quences of DNA with the ability to move from one site to another (see 16 Transposons and 17 Retroviruses and retroposons). An insertion usually abolishes the activity of a gene. Where such insertions have oc- curred, deletions of part or all of the inserted material, and sometimes of the adjacent regions, may subsequently occur. A significant difference between point mutations and the inser- tions/deletions is that the frequency of point mutation can be increased by mutagens, whereas the occurrence of changes caused by transpos- able elements is not affected. However, insertions and deletions can also occur by other mechanisms—for example, involving mistakes made during replication or recombination—although probably these are less common. And a class of mutagens called the acridines introduce (very small) insertions and deletions. 12 CHAPTER 1 Genes are DNA By Book_Crazy [IND]
  30. 30. 1.12 The effects of mutations can be reversed Key Concepts • Forward mutations inactivate a gene, and back mutations (or revertants) reverse their effects. • Insertions can revert by deletion of the inserted material, but deletions cannot revert. • Suppression occurs when a mutation in a second gene bypasses the effect of mutation in the first gene. Figure 1.22 shows that the isolation of revertants is an important" characteristic that distinguishes point mutations and insertions from deletions: • A point mutation can revert by restoring the original sequence or by gaining a compensatory mutation elsewhere in the gene. • An insertion of additional material can revert by deletion of the inserted material. • A deletion of part of a gene cannot revert. Mutations that inactivate a gene are called forward mutations. Their effects are reversed by back mutations, which are of two types. An exact reversal of the original mutation is called true reversion. So if an AT pair has been replaced by a GC pair, another mutation to restore the AT pair will exactly regenerate the wild-type sequence. Alternatively, another mutation may occur elsewhere in the gene, and its effects compensate for the first mutation. This is called second- site reversion. For example, one amino acid change in a protein may abolish gene function, but a second alteration may compensate for the first and restore protein activity. A forward mutation results from any change that inactivates a gene, whereas a back mutation must restore function to a protein damaged by a particular forward mutation. So the demands for back mutation are much more specific than those for forward mutation. The rate of back mutation is correspondingly lower than that of forward mutation, typi- cally by a factor of ~ 0. Mutations can also occur in other genes to circumvent the effects of mutation in the original gene. This effect is called suppression. A locus in which a mutation suppresses the effect of a mutation in another locus is called a suppressor. 1.13 Mutations are concentrated at hotspots Key Concepts ' The frequency of mutation at any particular base pair is determined by statistical fluctuation, except for hotspots, where the frequency is increased by at least an order of magnitude. So far we have dealt with mutations in terms of individual changes in the sequence of DNA that influence the activity of the genetic unit in which they occur. When we consider mutations in terms of the inactivation of the gene, most genes within a species show more or less similar rates of mutation relative to their size. This suggests that the gene can be regarded as a target for mutation, and that damage to The effects of mutations can be reversed SECTION 1.12 13 By Book_Crazy [IND]
  31. 31. any part of it can abolish its function. As a result, susceptibility to mu- tation is roughly proportional to the size of the gene. But consider the sites of mutation within the sequence of DNA; are all base pairs in a gene equally susceptible or are some more likely to be mutated than others? What happens when we isolate a large number of independent muta- tions in the same gene? Many mutants are obtained. Each is the result of an individual mutational event. Then the site of each mutation is deter- mined. Most mutations will lie at different sites, but some will lie at the same position. Two independently isolated mutations at the same site may constitute exactly the same change in DNA (in which case the same mutational event has happened on more than one occasion), or they may constitute different changes (three different point mutations are possible at each base pair). The histogram of Figure 1.23 shows the frequency with which mu- tations are found at each base pair in the lad gene of E. coli. The statis- tical probability that more than one mutation occurs at a particular site is given by random-hit kinetics (as seen in the Poisson distribution). So some sites will gain one, two, or three mutations, while others will not gain any. But some sites gain far more than the number of mutations ex- pected from a random distribution; they may have 10x or even 100x more mutations than predicted by random hits. These sites are called hotspots. Spontaneous mutations may occur at hotspots; and different mutagens may have different hotspots. 1.14 Many hotspots result from modified bases Key Concepts • A common cause of hotspots is the modified base 5-methylcytosine, which is spontaneously deaminated to thymine. Amajor cause of spontaneous mutation results from the presence of an unusual base in the DNA. In addition to the four bases that are inserted into DNA when it is synthesized, modified bases are some- times found. The name reflects their origin; they are produced by chem- ically modifying one of the four bases already present in DNA. The most common modified base is 5-methylcytosine, generated by a methylase enzyme that adds a methyl group to certain cytosine residues at specific sites in the DNA. Sites containing 5-methylcytosine provide hotspots for spontaneous point mutation in E. coli. In each case, the mutation takes the form of a GC to AT transition. The hotspots are not found in strains of E. coli that cannot methylate cytosine. The reason for the existence of the hotspots is that cytosine bases suffer spontaneous deamination at an appreciable frequency. In this re- action, the amino group is replaced by a keto group. Recall that deami- nation of cytosine generates uracil (see Figure 1.20). Figure 1.24 compares this reaction with the deamination of 5-methylcytosine where deamination generates thymine. The effect in DNA is to generate the base pairs GU and GT, respectively, where there is a mismatch be- tween the partners. All organisms have repair systems that correct mismatched base pairs by removing and replacing one of the bases. The operation of these systems determines whether mismatched pairs such as GU and GT result in mutations. 14 CHAPTER 1 Genes are DNA By Book_Crazy [IND]
  32. 32. Figure 1.25 shows that the consequences of deamination are different for 5-methylcytosine and cytosine. Deaminating the (rare) 5-methylcyto- sine causes a mutation, whereas deamination of the more common cyto- sine does not have this effect. This happens because the repair systems are much more effective in recognizing GU than G-T. E. coli contains an enzyme, uracil-DNA-glycosidase, that removes uracil residues from DNA (see 15.22 Base flipping is used by methylases and glycosylases). This action leaves an unpaired G residue, and a "re- pair system" then inserts a C base to partner it. The net result of these re- actions is to restore the original sequence of the DNA. This system protects DNA against the consequences of spontaneous deamination of cytosine (although it is not active enough to prevent the effects of the in- creased level of deamination caused by nitrous acid; see Figure 1.20). But the deamination of 5-methylcytosine leaves thymine. This creates" a mismatched base pair, G-T. If the mismatch is not corrected before the next replication cycle, a mutation results. At the next replication, the bases in the mispaired G-T partnership separate, and then they pair with new partners to produce one wild-type G-C pair and one mutant AT pair. Deamination of 5-methylcytosine is the most common cause of pro- duction of G-T mismatched pairs in DNA. Repair systems that act on G-T mismatches have a bias toward replacing the T with a C (rather than the alternative of replacing the G with an A), which helps to reduce the rate of mutation (see 15.24 Controlling the direction of mismatch re- pair). However, these systems are not as effective as the removal of U from GU mismatches. As a result, deamination of 5-methylcytosine leads to mutation much more often than does deamination of cytosine. 5-methylcytosine also creates hotspots in eukaryotic DNA. It is common at CpG dinucleotides that are concentrated in regions called CpG islands (see 21.19 CpG islands are regulatory targets). Although 5-methylcytosine accounts for - 1 % of the bases in human DNA, sites containing the modified base account for -30% of all point mutations. This makes the state of 5-methylcytosine a particularly important deter- minant of mutation in animal cells. The importance of repair systems in reducing the rate of mutation is emphasized by the effects of eliminating the mouse enzyme MBD4, a glycosylase that can remove T (or U) from mismatches with G. The re- sult is to increase the mutation rate at CpG sites by a factor of 3x. (The reason the effect is not greater is that MBD4 is only one of several sys- tems that act on G-T mismatches; we can imagine that elimination of all the systems would increase the mutation rate much more.) The operation of these systems casts an interesting light on the use of T in DNA compared with U in RNA. Perhaps it relates to the need of DNA for stability of sequence; the use of T means that any deaminations of C are immediately recognized, because they generate a base (U) not usually present in the DNA. This greatly increases the efficiency with which re- pair systems can function (compared with the situation when they have to recognize G-T mismatches, which can be produced also by situations where removing the T would not be the appropriate response). Also, the phosphodiester bond of the backbone is more labile when the base is U. 1.15 A gene codes for a single polypeptide Key Concepts • The one gene: one enzyme hypothesis summarizes the basis of modern genetics: that a gene is a stretch of DNA coding for a single polypeptide chain. • Most mutations damage gene function. A gene codes for a single polypeptide SECTION 1.15 15 By Book_Crazy [IND]
  33. 33. The first systematic attempt to associate genes with enzymes showed that each stage in a metabolic pathway is catalyzed by a single enzyme and can be blocked by mutation in a different gene. This led to the one gene: one enzyme hypothesis. Each metabolic step is cat- alyzed by a particular enzyme, whose production is the responsibility of a single gene. A mutation in the gene alters the activity of the protein for which it is responsible. A modification in the hypothesis is needed to accommodate proteins that consist of more than one subunit. If the subunits are all the same, the protein is a homomultimer, represented by a single gene. If the sub- units are different, the protein is a heteromultimer. Stated as a more general rule applicable to any heteromultimeric protein, the one gene: one enzyme hypothesis becomes more precisely expressed as one gene: one polypeptide chain. Identifying which protein represents a particular gene can be a pro- tracted task. The mutation responsible for creating Mendel's wrinkled- pea mutant was identified only in 1990 as an alteration that inactivates the gene for a starch branching enzyme! It is important to remember.that a gene does not directly generate a protein. As shown previously in Figure 1.2, a gene codes for an RNA, which may in turn code for a protein. Most genes code for proteins, but some genes code for RNAs that do not give rise to proteins. These RNAs may be structural components of the apparatus responsible for synthesizing proteins or may have roles in regulating gene expression. The basic principle is that the gene is a sequence of DNA that specifies the sequence of an independent product. The process of gene expres- sion may terminate in a product that is either RNA or protein. A mutation is a random event with regard to the structure of the gene, so the greatest probability is that it will damage or even abolish gene function. Most mutations that affect gene function are recessive: they represent an absence of function, because the mutant gene has been prevented from producing its usual protein. Figure 1.26 illus- trates the relationship between recessive and wild-type alleles. When a heterozygote contains one wild-type allele and one mutant allele, the wild-type allele is able to direct production of the enzyme. The wild- type allele is therefore dominant. (This assumes that an adequate amount of protein is made by the single wild-type allele. When this is not true, the smaller amount made by one allele as compared to two alleles results in the intermediate phenotype of a partially dominant allele in a heterozygote.) 1.16 Mutations in the same gene cannot complement Key Concepts • A mutation in a gene affects only the protein coded by the mutant copy of the gene, and does not affect the protein coded by any other allele. * Failure of two mutations to complement (produce wild-phenotype) when they are present in trans configuration in a heterozygote means that they are part of the same gene. How do we determine whether two mutations that cause a similar phenotype lie in the same gene? If they map close together, they may be alleles. However, they could also represent mutations in two dif- 16 CHAPTER 1 Genes are DNA By Book_Crazy [IND]
  34. 34. ferent genes whose proteins are involved in the same function. The complementation test is used to determine whether two mutations lie in the same gene or in different genes. The test consists of making a het- erozygote for the two mutations (by mating parents homozygous for each mutation). If the mutations lie in the same gene, the parental genotypes can be represented as The first parent provides an ml mutant allele and the second parent provides an m-i allele, so that the heterozygote has the constitution No wild-type gene is present, so the heterozygote has mutant pheno- type. If the mutations lie in different genes, the parental genotypes can be represented as Each chromosome has a wild-type copy of one gene (represented by the plus sign) and a mutant copy of the other. Then the heterozygote has the constitution in which the two parents between them have provided a wild-type copy of each gene. The heterozygote has wild phenotype; the two genes are said to complement. The complementation test is shown in more detail in Figure 1.27. The basic test consists of the comparison shown in the top part of the figure. If two mutations lie in the same gene, we see a difference in the phenotypes of the trans configuration and the cis configuration. The trans configuration is mutant, because each allele has a (different) mutation. But the cis configuration is wild-type, because one allele has two mutations but the other allele has no mutations. The lower part of the figure shows that if the two mutations lie in different genes, we always see a wild phenotype. There is always one wild-type and one mutant allele of each gene, and the configuration is irrelevant. Failure to complement means that two mutations are part of the same genetic unit. Mutations that do not complement one another are said to comprise part of the same complementation group. An- other term that is used to describe the unit defined by the comple- mentation test is the cistron. This is the same as the gene. Basically these three terms all describe a stretch of DNA that functions as a unit to give rise to an RNA or protein product. The properties of the gene with regards to complementation are explained by the fact that this prod- uct is a single molecule that behaves as a functional unit. double mutant Mutations in the same gene cannot complement SECTION 1.16 17 By Book_Crazy [IND]
  35. 35. 1.17 Mutations may cause loss-of-function or gain-of-function Key Concepts • Recessive mutations are due to loss-of-function by the protein product. • Dominant mutations result from a gain-of-function. • Testing whether a gene is essential requires a null mutation (one that completely eliminates its function). • Silent mutations have no effect, either because the base change does not change the sequence or amount of protein, or because the change in protein sequence has no effect. • Leaky mutations do affect the function of the gene product, but are not revealed in the phenotype because sufficient activity remains. Figure 1.28 Mutations that do not affect protein sequence or function are silent. Mutations that abolish all protein activity are null. Point mutations that cause loss-of-function are recessive; those that cause gain-of-function are dominant. The various possible effects of mutation in a gene are summarized in Figure 1.28. When a gene has been identified, insight into its func- tion in principle can be gained by generating a mutant or- ganism that entirely lacks the gene. A mutation that completely eliminates gene function, usually because the gene has been deleted, is called a null mutation. If a gene is essential, a null mutation is lethal. To determine what effect a gene has upon the pheno- type, it is essential to characterize a null mutant. When a mutation fails to affect the phenotype, it is always possi- ble that this is because it is a leaky mutation—enough active product is made to fulfill its function, even though the activity is quantitatively reduced or qualitatively dif- ferent from the wild type. But if a null mutant fails to af- fect a phenotype, we may safely conclude that the gene function is not necessary. Null mutations, or other mutations that impede gene function (but do not necessarily abolish it entirely) are called loss-of-function mutations. A loss-of-function mutation is recessive (as in the example of Figure 1.26). Sometimes a mutation has the opposite effect and causes a protein to acquire a new function; such a change is called a gain-of-function mutation. A gain-of-function mutation is dominant. Not all mutations in DNA lead to a detectable change in the phenotype. Mutations without apparent effect are called silent mutations. They fall into two types. Some involve base changes in DNA that do not cause any change in the amino acid present in the corre- sponding protein. Others change the amino acid, but the replacement in the protein does not affect its activity; these are called neutral substitutions. 18 CHAPTER 1 Genes are DNA By Book_Crazy [IND]

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