Molecular biology d clark (elsevier, 2005)

MOLECULAR BIOLOGY David Clark Southern Illinois University AMSTERDAM • BOSTON • HEIDELBERG • LONDON NEW YORK • OXFORD • PARIS • SAN DIEGO SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO MOLECULAR BIOLOGY Senior Acquisitions Editor Project Manager Editorial Assistant Marketing Manager Cover Design Composition Cover Printer Interior Printer Jeremy Hayhurst Kyle Sarofeen Desiree Marr Linda Beattie Eric DeCicco SNP Best-set Typesetter Ltd., Hong Kong Hing Yip Printing Co., Ltd Hing Yip Printing Co., Ltd Elsevier Academic Press 30 Corporate Drive, Suite 400, Burlington, MA 01803, USA 525 B Street, Suite 1900, San Diego, California 92101-4495, USA 84 Theobald’s Road, London WC1X 8RR, UK This book is printed on acid-free paper Copyright © 2005, Elsevier Inc All rights reserved No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone: (+44) 1865 843830, fax: (+44) 1865 853333, e-mail: permissions@elsevier.co.uk You may also complete your request on-line via the Elsevier homepage (http://elsevier.com), by selecting “Customer Support” and then “Obtaining Permissions.” Library of Congress Cataloging-in-Publication Data Application submitted British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library ISBN: 0-12-175551-7 For all information on all Elsevier Academic Press Publications visit our Web site at www.books.elsevier.com Printed in China 05 06 07 08 09 10 Dedication This book is dedicated to Lonnie Russell who was to have been my coauthor A few months after we started this project together, in early July 2001, Lonnie drowned in the Atlantic Ocean off the coast of Brazil in a tragic accident Preface This book’s subtitle, Understanding the Genetic Revolution, reflects the massive surge in our understanding of the molecular foundations of genetics in the last fifty years In the next half century our understanding of how living organisms function at the molecular level, together with our ability to intervene, will expand in ways we are only just beginning to perceive Today we now know that genes are much more than the abstract entities proposed over a century ago by Mendel Genes are segments of DNA molecules, carrying encoded information Indeed, genes have now become chemical reagents, to be manipulated in the test tube In the days of classical genetics genes represented inherited characteristics but were themselves inviolate, rather like atoms before the twentieth century Today both genes and atoms have sub-components to be tinkered with A full understanding of how living organisms function includes an appreciation of how cells operate at the molecular level This is of vital importance to all of us as it becomes ever more clear that molecular factors underlie many health problems and diseases While cancer is the “classic” case of a disease that only became understandable when its genetic basis was revealed, it is not the only one by any means Today the molecular aspects of medicine are expanding rapidly and it will soon be possible to personally tailor clinical treatment by taking into account the genetic make-up of individual patients Rather than attempting to summarize my view of modern molecular biology (the book itself, I hope, accomplishes that) in a short preface, I’d like to briefly address what this book is not It is not intended as a reference work for faculty or researchers but rather as a survey-oriented textbook for upper division students in a variety of biological sub-disciplines In particular it is intended for final year undergraduates and beginning graduate students This book does not attempt to be exhaustive in its coverage, even as a textbook There is a second book in this series (Biotechnology: Applying the Genetic Revolution, 2006) co-authored with Nanette Pazdernik, which essentially picks up where this book ends Both books, I hope, effectively survey the foundations and applications of modern molecular genetics Many, perhaps most, of the students using this book will be well versed in the basics of modern genetics and cell biology and so can pick and choose from the topics covered as needed However, others will not be so well prepared, due in part to the continuing influx into molecular biology of students from related disciplines For them I’ve tried to create a book whose early chapters cover the basics, before launching out into the depths Because of the continuing interest in applying molecular biology to an ever widening array of topics, I have tried to avoid overdoing detail (depth) in favour of breadth This in no way minimizes the importance of the subject matter for cell biologists but instead emphasizes that molecular biology is applicable to more than just human medicine and health The genetic revolution has also greatly impacted other important areas such as agriculture, veterinary medicine, animal behaviour, evolution, and microbiology Students of these, and related disciplines, all need to understand molecular biology at some level Finally there are no references or extra reading at the ends of the chapters, for two reasons My own cross-questioning has revealed that neither myself nor most of my colleagues and students have ever actually used such textbook references just as we rarely watch the extra material on DVDs providing actors’ insights, extra scenes, and outtakes The student has enough to deal with in the core material Secondly, anyone who wants up-to-date reference material is far better advised to run a web search PubMed, Google Scholar and Scirus.com are good choices Feedback (hopefully positive!) is welcome David Clark, Carbondale, Illinois, January 2005 Acknowledgements I would like to thank the following individuals for their help in providing information, suggestions for improvement and encouragement: Laurie Achenbach, Rubina Ahsan, Phil Cunningham, Michelle McGehee, Donna Mueller, Dan Nickrent, Joan Slonczewski Especial thanks go to Nanette Pazdernik for help in editing many of the chapters and to Karen Fiorino for creating most of the artwork vii Introduction Molecular Genetics Is Driving the Biotechnology Revolution Although the breeding of plants and animals goes back thousands of years, only in the last couple of centuries has genetics emerged as a field of scientific study Classical genetics emerged in the 1800s when the inheritance patterns of such things as hair or eye color were examined and when Gregor Mendel performed his famous experiments on pea plants Techniques revealing how the inherited characteristics that we observe daily are linked to their underlying biochemical causes have only been developed since World War II The resulting revelation of the molecular basis of inheritance has resulted in the increasing use of the term “molecular.” Often the term “molecular biology” refers to the biology of those molecules related to genes, gene products and heredity—in other words, the term molecular biology is often substituted for the perhaps more appropriate term, molecular genetics A more broad-minded definition of molecular biology includes all aspects of the study of life from a molecular perspective Although the molecular details of muscle operation or plant pigment synthesis could be included under this definition, in practice, textbooks are limited in length In consequence, this book is largely devoted to the molecular aspects of the storage and transmission of biological (i.e., genetic) information Although there is great diversity in the structures and lifestyles of living organisms, viewing life at the molecular level emphasizes the inherent unity of life processes Perhaps it is this emergent unity, rather than the use of sophisticated molecular techniques, that justifies molecular biology as a discipline in its own right Instead of an ever-expanding hodge-podge of methods for analyzing different organisms in more and more detail, what has been emerged from molecular analysis is an underlying theme of information transmission that applies to all life forms despite their outward differences Society is in the midst of two scientific revolutions One is in the realm of technology of information, or computers, and the other in molecular biology Both are related to the handling of large amounts of encoded information In one case the information is man made, or at any rate man-encoded, and the mechanisms are artificial; the other case deals with the genetic information that underlies life Biology has reached the point where the genes that control the makeup and functioning of all living creatures are being analyzed at the molecular level and can be altered by genetic engineering In fact, managing viii and analyzing the vast mass of genetic information constantly emerging from experimentation requires the use of sophisticated software and powerful computers The emerging information revolution rivals the industrial revolution in its importance, and the consequences of today’s findings are already changing human lives and will continue to alter the lives of future generations Data is accumulating about the molecules of inheritance and how they are controlled and expressed at an ever faster and faster pace This is largely due to improved techniques, such as PCR (polymerase chain reaction; see Ch 23) and DNA (deoxyribonucleic acid) arrays (see Ch 25) In particular, methods have recently been developed for the rapid, simultaneous and automated analysis of multiple samples and/or multiple genes One major impact of molecular biology is in the realm of human health The almost complete sequence of the DNA molecules comprising the human genome was revealed in the year 2003 So, in theory, science has available all of the genetic information needed to make a human being However, the function of most of a human’s approximately 35,000 genes remains a mystery Still more complex is the way in which the expression of these genes is controlled and coordinated Inherited diseases are due to defective versions of certain genes or to chromosomal abnormalities To understand why defective genes cause problems, it is important to investigate the normal roles of these genes As all disease has a genetic component, the present trend is to redefine physical and mental health from a genetic perspective Even the course of an infectious disease depends to a significant extent on built-in host responses, which are determined by host genes For example, humans with certain genetic constitutions are at much greater risk than others of getting SARS, even though this is an emerging disease that only entered the human population in the last few years The potential is present to improve health and to increase human and animal life spans by preventing disease and slowing the aging process Clinical medicine is changing rapidly to incorporate these new findings The other main arena where biotechnology will have a massive impact is agriculture New varieties of genetically engineered plants and animals have already been made and some are in agricultural use Animals and plants used as human food sources are being engineered to adapt them to conditions which were previously unfavorable Farm animals that are resistant to disease and crop plants that are resistant to pests are being developed in order to increase yields and reduce costs The impact of these genetically modified organisms on other species and on the environment is presently a controversial issue Table of Contents CHAPTER CHAPTER CHAPTER CHAPTER CHAPTER CHAPTER CHAPTER CHAPTER CHAPTER CHAPTER 10 CHAPTER 11 CHAPTER 12 CHAPTER 13 CHAPTER 14 CHAPTER 15 CHAPTER 16 CHAPTER 17 CHAPTER 18 CHAPTER 19 CHAPTER 20 CHAPTER 21 CHAPTER 22 CHAPTER 23 CHAPTER 24 CHAPTER 25 CHAPTER 26 Basic Genetics Cells and Organisms 21 DNA, RNA and Protein 51 Genes, Genomes and DNA 75 Cell Division and DNA Replication 103 Transcription of Genes 132 Protein Structure and Function 154 Protein Synthesis 197 Regulation of Transcription in Prokaryotes 234 Regulation of Transcription in Eukaryotes 262 Regulation at the RNA Level 281 Processing of RNA 302 Mutations 333 Recombination and Repair 368 Mobile DNA 396 Plasmids 425 Viruses 453 Bacterial Genetics 484 Diversity of Lower Eukaryotes 508 Molecular Evolution 533 Nucleic Acids: Isolation, Purification, Detection, and Hybridization 567 Recombinant DNA Technology 599 The Polymerase Chain Reaction 634 Genomics and DNA Sequencing 662 Analysis of Gene Expression 693 Proteomics: The Global Analysis of Proteins 717 Glossary 745 Index 771 ix Detailed Contents CHAPTER Basic Genetics Gregor Mendel Was the Father of Classical Genetics Genes Determine Each Step in Biochemical Pathways Mutants Result from Alterations in Genes Phenotypes and Genotypes Chromosomes Are Long, Thin Molecules That Carry Genes Different Organisms may Have Different Numbers of Chromosomes Dominant and Recessive Alleles Partial Dominance, Co-Dominance, Penetrance and Modifier Genes Genes from Both Parents Are Mixed by Sexual Reproduction Sex Determination and Sex-Linked Characteristics Neighboring Genes Are Linked during Inheritance Recombination during Meiosis Ensures Genetic Diversity Escherichia coli Is a Model for Bacterial Genetics CHAPTER Cells and Organisms What Is Life? Living Creatures Are Made of Cells Essential Properties of a Living Cell Prokaryotic Cells Lack a Nucleus Eubacteria and Archaebacteria Are Genetically Distinct Bacteria Were Used for Fundamental Studies of Cell Function Escherichia coli (E coli) Is a Model Bacterium Where Are Bacteria Found in Nature? Some Bacteria Cause Infectious Disease, but Most Are Beneficial Eukaryotic Cells Are Sub-Divided into Compartments The Diversity of Eukaryotes Eukaryotes Possess Two Basic Cell Lineages Organisms Are Classified x 11 13 Some Widely Studied Organisms Serve as Models Yeast Is a Widely Studied Single-Celled Eukaryote A Roundworm and a Fly are Model Multicellular Animals Zebrafish are used to Study Vertebrate Development Mouse and Man Arabidopsis Serves as a Model for Plants Haploidy, Diploidy and the Eukaryote Cell Cycle Viruses Are Not Living Cells Bacterial Viruses Infect Bacteria Human Viral Diseases Are Common A Variety of Subcellular Genetic Entities Exist 16 17 21 22 23 23 27 28 29 31 32 34 34 36 36 38 40 41 42 44 44 45 46 47 48 49 CHAPTER DNA, RNA and Protein 15 40 Nucleic Acid Molecules Carry Genetic Information Chemical Structure of Nucleic Acids DNA and RNA Each Have Four Bases Nucleosides Are Bases Plus Sugars; Nucleotides Are Nucleosides Plus Phosphate Double Stranded DNA Forms a Double Helix Base Pairs are Held Together by Hydrogen Bonds Complementary Strands Reveal the Secret of Heredity Constituents of Chromosomes The Central Dogma Outlines the Flow of Genetic Information Ribosomes Read the Genetic Code The Genetic Code Dictates the Amino Acid Sequence of Proteins Various Classes of RNA Have Different Functions Proteins, Made of Amino Acids, Carry Out Many Cell Functions The Structure of Proteins Has Four Levels of Organization Proteins Vary in Their Biological Roles 51 52 52 54 55 56 57 59 60 63 65 67 69 70 71 73 770 Glossary uracil (U) A pyrimidine base found in RNA that may pair with adenine uracil-N-glycosylase Enzyme that removes uracil from DNA urea A nitrogen waste product of animals; also widely used as a denaturant of proteins uridine The nucleoside consisting of uracil plus ribose urkaryote Hypothetical ancestor that provided the genetic information of the eukaryotic nucleus U-RNA Uracil-rich small RNA (includes snRNA and snoRNA) vaccination Artificial induction of the immune response by injecting foreign proteins or other antigens variable number tandem repeats (VNTR) Cluster of tandemly repeated sequences in the DNA, whose number of repeats differs from one individual to another variant surface glycoprotein (VSG) Glycoprotein found on surface of trypanosomes that is encoded by multiple gene copies and varied to avoid recognition by the animal immune system vector (a) In molecular biology a vector is molecule of DNA which can replicate and is used to carry cloned genes or DNA fragments; (b) in general biology a vector is an organism (such as a mosquito) that carries and distributes a disease-causing microorganisms (such as yellow fever or malaria) vegetative reproduction Form of reproduction in which there is no reshuffling of the genes between two individuals (same as asexual reproduction) vertical gene transfer Transfer of genetic information from an organism to its descendents viral genome Molecule of DNA or RNA that carries the genes of a virus virion Virus particle viroid Naked single-stranded circular RNA that forms a stable highly base-paired rod-like structure and replicates inside infected plant cells Viroids not encode any proteins but possess self-cleaving ribozyme activity virulence factors Proteins that promote virulence in infectious bacteria Include toxins, adhesins and proteins protecting bacteria from the immune system virulence plasmid Plasmid that carries genes for virulence factors that play a role in bacterial infection virus Subcellular parasite with genes of DNA or RNA which replicates inside the host cell upon which it relies for energy and protein synthesis In addition, it has an extracellular form, in which the virus genes are contained inside a protective coat virusoid Parasitic RNA molecule that does not encode any proteins but depends on a helper virus for replication and capsid formation Vmax Maximum velocity of an enzyme VNTR See variable number tandem repeats Western blotting Detection technique in which a probe, usually an antibody, binds to a protein target molecule wild-type The original or “natural” version of a gene or organism wobble rules Rules allowing less rigid base pairing but only for codon/anticodon pairing writhe Same as writhing number, W writhing number, W The number of supercoils in a molecule of DNA (or double-stranded RNA) X-chromosome Female sex chromosome; possession of two X-chromosomes causes female gender in mammals X-gal (5-bromo-4-chloro-3-indolyl b-D-galactoside) Artificial substrate that is split by b-galactosidase, releasing a blue dye X-inactivation The condensation and complete shutting down of gene expression of one of the two X-chromosomes in cells of female mammals Xis protein Enzyme that reverses DNA integration by removing a segment of dsDNA and resealing the gap leaving behind an intact recognition sequence Same as excisionase Not to be confused with Xist RNA involved in X chromosome silencing Xist gene A gene that causes the inactivation of the Xchromosome that carries it X-phos 5-bromo-4-chloro-3-indolyl phosphate, an artificial substrate that is split by alkaline phosphatase, releasing a blue dye Y-chromosome Male sex chromosome; possession of a Y-chromosome plus an X-chromosome causes male gender in mammals yeast artificial chromosome (YAC) Single copy vector based on yeast chromosome that can carry very long inserts of DNA Widely used in the human genome project Y-guy Hypothetical male human ancestor thought to have lived in Africa around 100,000–200,000 years ago Z-DNA An alternative form of DNA double helix with left-handed turns and 12 base pairs per turn Z-form An alternative form of double helix with lefthanded turns and 12 base pairs per turn Both DNA and dsRNA may be found in the Z-form zinc finger One type of DNA-binding motif common in proteins Zoo blotting Comparative Southern blotting using DNA target molecules from several different animals to test whether the probe DNA is from a coding region zwitterion Same as dipolar ion; a molecule with both a positive and a negative charge zygote Cell formed by union of sperm and egg which develops into a new individual Index Note: Italicized page numbers refer to illustrations, tables, and notes Numbers 2-micron plasmid, 450–51 3-alkyl adenine, 349 3¢-exonuclease, 112 3¢ splice site, 310 3¢-untranslated region (3¢-UTR), 136 4-methylumbelliferyl phosphate, 696 5-methyl-cytosine, 357 5¢ splice site, 310 5¢-terminal oligopyrimidine tract (5¢-TOP), 290–91 5¢-untranslated region (5¢-UTR), 135, 209, 285 7SL RNA gene, 415 -10 region, 136 30 nanometer fiber, 96 30S initiation complex, 211, 212 30S subunit, 204 -35 region, 136 40S subunit, 204 50S subunit, 204 60S subunit, 204 70S initiation complex, 211, 212 70S ribosome, 206 80S ribosome, 204 A A (acceptor) site, 211 abortive transposition, 410 acceptor (A) site, 211 acceptor stem, 201 Ac element, 411 acetosyringone, 448 acetylases, 182 acetylation, 271 aconitase, 285–86 acquired immunodeficiency syndrome (AIDS), 472 acridine orange, 348 activation energy, 182–84 activators, 140–43 functions of, 243–44 modification of, 252 and repressors, 246–48 active sites, 73, 174 acyl phosphates, 538–39 Ada protein, 386–87 adenine, 54–56, 60, 349 adenosine monophosphate (AMP), 55 adenoviruses, 464 adhesins, 446–47 A-DNA, 92–93 Aequorea victoria, 699 African Eve hypothesis, 560–62 agar, 30 agarose, 570 agarose gel electrophoresis, 570–72 Agrobacterium, 447–50, 515 AIDS (acquired immunodeficiency syndrome), 472 alanine, 347 aldose reductase, 179 alkaline phosphatase, 588, 696, 697 alleles, co-dominance in, 10 dominant, 8–9 non-coding DNA, 78 partial dominance in, 9–10 penetrance in, 10–11 recessive, 8–9 allo-lactose, 248 allosteric enzymes, 187–89 allosteric proteins, 145, 189 alpha carbon, 70, 155, 158–60 alpha complementation, 612, 614 alpha fragment, 612 alpha helix, 160–63 alpha-hemolysin, 676 alpha-particles, 350–51 alternative sigma factors, 238–42 alternative splicing, 198, 315–18 Alu element, 83, 415, 416 amber, 363 Ames test, 363–64 amethopterin, 109 amikacin, 441 amino acids, 155; see also proteins alignment of related sequences, 546 asymmetry around alpha-carbon, 158–60 charging transfer RNA (tRNA) with, 204, 205 conservative substitution of, 337 essential, 517 in formation of biological peptides, 155–58 general features of, 70 general structure of, 156 genetic code, 67–68 hydrogen bonds, 165 L- and D- forms of, 159 polymerization of, 538 post-translational modifications, 227 properties of, 159 in protein synthesis, 65 radical replacement of, 337–38 aminoacyl tRNA synthetases, 204 aminoglycosides, 230, 440–41 AMP (adenosine monophosphate), 55 amp gene, 610 ampicillin, 438–39, 610 anaerobic respiration, 252 analogs, 184–85 anchor sequences, 649 ancient DNA, 562–64 aneuploid, animals, 39 annealing, 100–102 Annelida, 39–40 antennapedia genes, 530–32 anti-anti-sigma factors, 242 antibiotics, 33 aminoglycosides, 440–41 beta-lactam, 438–39 chloramphenicol, 439–40 in inhibition of protein synthesis, 230–31 resistance to, 364, 436–38, 695 sulfonamides, 442 tetracyclines, 441–42 trimethoprim, 442 antibodies, 623 anticodon loop, 201 anticodons, 67, 200 antifreeze proteins, 174 antisense RNA, 69, 288–90, 432–35 anti-sense strands, see template strands anti-Shine-Dalgarno sequence, 210–11 anti-sigma factors, 242–43 anti-termination factors, 258–61 anti-terminator proteins, 237 AP endonuclease, 384 aphids, 517 Apicomplexa, 513–14 apicoplasts, 514 apolipoprotein B100, 324–25 apoprotein, 170 apoptosis, 42 AP-site, 384 araBAD operon, 248 Arabidopsis thaliana, 44–45, 450 arabinose, 167, 248 AraC protein, 248, 250 Archaea, 555, 557 archaebacteria, 28–29; see also bacteria; eubacteria classification of, 38 vs eubacteria, 554–55 gene transfer in, 504–6 histones in, 98 lipids of, 29 ARE-binding protein, 330 771 772 Index arginine, 251 ArgR repressor, 251 Arthropoda, 39–40 artificial chromosomes, 620–21 Ascomycete, 394–95 ascospores, 395, 527 ascus, 395, 527 asexual reproduction, 104, 485 aspartase, 179–80 Aspergillus nidulans, 128 Aspergillus oryzae, 129, 707 atmosphere, 534–35 ATPase, 182 attachment sites, 374, 460, 494 attenuation, 297–99 attenuation protein, 299 autogenous regulation, 248 autoradiography, 584–85 autotrophic theory, 544–45 auxin, 448 Avery, Oswald, 76, 488–89 avidin, 587–88, 726 B B1 element, 83 Bacillus, spore formation in, 239–42 Bacillus sphaericus, 564 BAC (bacterial artificial chromosome), 620–21, 681, 683 bacteria, 23 antibiotic resistance of, 436–38 aminoglycosides, 440–41 beta-lactam antibiotics, 438–39 chloramphenicol, 439–40 sulfonamides, 442 tetracyclines, 441–42 trimethoprim, 442 archaebacteria, 28–29 cell division in, 124 components of, 27 coupled transcription-translation in, 216–17 disease-causing, 34 DNA supercoiling, 88–89 eubacteria, 28 exponential growth of culture, 30 gram-negative, 501–3 gram-positive, 501–4 habitats, 32–33 immune systems, 445–46 replication in, 124–25 ribosomal proteins, 286–87 in studies of cell function, 29–30 bacterial artificial chromosome (BAC), 620–21, 681, 683 bacterial genetics, 485 Archaebacteria, 504–6 circular DNA, gene sequencing, 506–7 homologous recombination, 485–87 reproduction vs gene transfer, 485 transduction, 493–95 transfection, 489–90 transfer of chromosomal genes, 496–501 transfer of plasmids, 495–96 transformation, 487–88, 491–93 bacteriocins, 33, 444–46 bacterioferritin, 284 bacteriophage G, 462 bacteriophage M13, 465, 668–69 bacteriophage Mu, 417–20 bacteriophages, 458–60; see also viruses double-stranded DNA, 465 entry into cells, 460, 461 estimated number of, 459 with heads and tails, 466 infection of bacteria by, 47–48 isolation of, 459 resistance to, 365 RNA-containing, 469 single-stranded DNA, 463–64 transduction, 493–95 as vectors, 616–18 bacteriophage T4, 458 bacteriophage T7, 633 BamHI restriction enzyme, 605–6 bandshift assay, 703 Barr bodies, 278 base, 52 base analogs, 348 base pairs, 57–59 base substitution mutations, 336, 360 Bdellovibrio bacterivorus, 34 B-DNA, 92 Beadle, G.W., bent DNA, 87–88 Bernstein, Harris, 361 beta-bend, 162–63 beta-galactosidase, 178; see also enzymes blue/white screening for, 615 in detection of insertions in vectors, 612 encoding by lacZ gene, 245 measuring the level of, 186 in monitoring gene expression, 696 substrates used by, 697 beta-lactam antibiotics, 438–39 beta-lactamase, 173 functions of, 182 resistance plasmids, 438–39, 610 beta-mercaptoethanol (BME), 196 beta-particles, 350–51 beta sheet, 160–63 binary fission, 485 binding proteins, 174 biochemical pathways, bioinformatics, 690–92 biological classification, 38–40 biopanning, 731 for RNA-binding proteins, 733 in screening phage display libraries, 732 biotin, 587–88, 713 bla gene, 610 blotting, 592–95, 722, 724 blue/white screening, 612, 615 blunt ends, 603 BME (beta-mercaptoethanol), 196 Borrelia, 429 boundary elements, 265 Brachydanio rerio, 42–44 Bragg, William, 77 branch site, 310 bromouracil, 349 Buchnera, 517 budding, 41 C Caedibacter, 515–16 Caenorhabditis elegans, 41–42 mutation rate in, 359 number of genes in, 685 RNA interference in, 293–94 Calico cat, 280 calmodulin, 741 capping, 306–8 capsids, 454 carboxypetidase, 181 carboxy-terminal domain (CTD), 150 carrier proteins, 174–75 CAT (chloramphenicol acetyl transferase), 440 catenanes, 91 cauliflower mosaic virus, 466–67 CBP (chitin-binding protein), 729–30 cccDNA (covalently closed circular DNA), 89 cDNA (complementary DNA), 415; see also DNA cloning, 624–26 synthesis of, 477, 646 Celera Genomics, 681 cell cycle, 131 cell division, 104 in bacteria, 124 in higher organisms, 130–31 cells, 23 differentiation, 24 essential properties of, 23–26 eukaryotic, 26, 35–36 prokaryotic, 27 cellular recognition, 173 central dogma, 63 centrifugation, 568 centromeres, 62, 84 centromere sequences, 84, 615–16 cephalosporins, 439 cesium chloride, 85 CG-islands, 274 chain termination mutations, 338–40 chain termination sequencing, 663–68 chaotropic agents, 195 chaperonins, 176–77 heat shock proteins (HSPs), 238 in protein folding, 224–25 checkerboard diagrams, 13 chelating agents, 568 Index chemical extraction, 568 chemical mutagens, 348–50, 363–64 chemiluminescence, 588 chimera, 600 chiral center, 158 chi sites, 373 chitin-binding protein (CBP), 729–30 Chlamydomonas, 511 chloramphenicol, 230, 439–40 chloramphenicol acetyl transferase (CAT), 440 chlorophyll, 36 chloroplast polyadenylate binding protein (cPABP), 287 chloroplasts, 36 genome of, 510–11 protein synthesis in, 225–26 chordata, 40 chromatids, 98 chromatin, 60, 95 chromatin remodeling complexes, 272 chromogenic substrates, 185 chromosomes, 6–7 artificial, 620–21 banding patterns, 63–64 eukaryotic, 129–30 genetic information in, 61 homologous, interphase, 99 metaphase, 99 replication of, 118–20 separation of, 122–24 structural components of, 60 chromosome walking, 626–29 chymotrypsin, 180 ciliates, 517–20 ciprofloxacin, 90 cI protein, 633 cistrons, 134, 215–16 clamp-loading complex, 111 classes, 40 clavulanic acid, 438–39 cloning, 600 chromosome walking, 626–28, 629 complementary DNA (cDNA), 624– 26 cutting of DNA by restriction enzymes, 602–3 gene libraries, 621–23 joining DNA fragments, 603–4 recognition of DNA, 601 restriction and modification of DNA, 600–601 restriction fragment polymorphisms (RFLPs), 607–8, 609 restriction map, 604–7 by subtractive hybridization, 628–31 vectors, 608 Clontech Corp., 699 cloverleaf structure, 200–201 Cnidaria, 39–40 coconut cadang-cadang viroid, 481 coding RNA, 304 coding strands, 133 co-dominance, 10 codons, 67, 199–200 cofactors, 71, 160, 169–72 co-immunoprecipitation, 737–40 cointegrate, 404 ColE plasmids, 444, 610 colicins, 443–44; see also plasmids immunity system, 445 modes of action, 444–45 plasmids, 610 synthesis and release of, 446 colonization factors, 446–47 competence pheromones, 491 competent cells, 487, 489 competitive inhibitors, 184 complementary DNA (cDNA), 415; see also DNA cloning, 624–26 synthesis of, 477, 646 complementary sequences, 59–60 complex transposons, 402 composite transposons, 406–8; see also transposons evolution of, 409 principle of, 408 conditional mutations, 338 conjugated proteins, 172 conjugation, 485 bacterial, 497 time of entry by, 501 conjugation bridge, 496, 497 conjugative transposons, 420–21, 504 consensus sequence, 81–82 conservative substitution, 337 conservative transposition, 401; see also transpositions movement by, 403 outline of, 402 vs replicative transposition, 406–7 constitutive genes, 140 contigs, 680 assembling genome from, 683 closing gaps between, 682 controlled pore glass (CPG), 575 copy number, 8, 427, 432–35 core enzyme, 111 co-repressors, 250, 272 corn, 412 Corynebacterium diptheriae, 228 cosmid vectors, 617–19 cos sequences, 616 cotransfer frequency, 500 cotranslational export, 223 coupled transcription-translation, 216–17 covalently closed circular DNA (cccDNA), 89 cowpox virus, 468 cPABP (chloroplast polyadenylate binding protein), 287 CPG (controlled pore glass), 575 773 Crick, Francis, 58, 76–77 cristae, 35 Cro protein, 193 crossover, 16–17, 369, 371 crossover resolvase, 122 crown gall disease, 447 Crp protein, 255, 256 cruciform structures, 91–92 cryptic plasmids, 427 CsrAB regulatory system, 283 CsrA protein, 283 CsrB RNA, 283 CTD (carboxy-terminal domain), 150 C-terminus, 71, 155 cut-and-paste transposition, 401 cyclic AMP receptor protein, 255, 256 cycloheximide, 230 CYP2D6 isoenzyme, 688 cysteine, 166, 229 cystine, 167 cytochrome c, 546, 551–52 cytochrome P450, 688 cytokinesis, 130 cytokinin, 448 cytoplasm, 25 cytosine, 54–56, 60, 349, 357 cZipcode sequence, 688 D Dam (DNA adenine methylase), 380 Danio rerio, 42–44 data mining, 690 daughter chromosomes, 61, 122–24 Dcm (DNA cytosine methylase), 380 deaminase, 324 deamination, 353, 357 defective interfering RNA (DI-RNA), 479 degenerate primers, 640–41 deletion mutations, 335, 340–41 demethylases, 274 denaturants, 196 denaturation, 100, 194–96, 591 denaturing gradient gel electrophoresis (DGGE), 573–74 de novo methylase, 274 deoxy-NTPs (deoxyribonucleoside 5¢triphosphates), 109, 120–21 deoxynucleosides, 55 deoxynucleotides, 55 deoxyribonuclease I (Dnase I), 705–6 deoxyribonucleases, 600 deoxyribonucleic acid, see DNA deoxyribonucleoside 5¢-triphosphates (deoxy-NTPs), 109, 120–21 deoxyribose, 52, 54, 666 deoxyuridine triphosphate (deoxyUTP), 587 deoxyUTP (deoxyuridine triphosphate), 587 detergents, 195, 568 deuterostomes, 532 774 Index DGGE (denaturing gradient gel electrophoresis), 573–74 DHF (dihydrofolate), 109 Dicer, 292 dideoxynucleotides, 665–68 dideoxyribose, 665, 666 dideoxy sequencing, 663–68 differential display PCR, 647–48 differentiation, 23 dif sites, 122 digoxigenin, 587 dihydrofolate (DHF), 109 dihydrofolate reductase, 109 dimethoxytrityl (DMT) group, 575 diphthamide, 227–28 diphtheria, 228 diploidy, 7, 29, 45, 277 dipolar ions, 155 directed mutagenesis, 366–67, 651 DI-RNA (defective interfering RNA), 479 disulfide bonds, 166 dmd gene, 628 DMT (dimethoxytrityl) group, 575 DNA adenine methylase (Dam), 380 DnaA protein, 118–19 DNA-binding proteins, 190–94 DnaB protein, 107 DNA chips, 672–73, 710 DNA cytosine methylase (Dcm), 380 DNA (deoxyribonucleic acid), 6–7; see also nucleic acids; RNA alternative helical structures, 92–95 annealing of, 100–102 base pairs, 57–59 bases, 54–56 blotting, 592–95 chemical structure of, 52 chemical tagging of, 587–88 complementary sequences, 59–60 cutting of, 602–3 detection of, 583–87 autoradiography, 584–85 fluorescence, 585–87 scintillation counting, 583–84 double-helix structure of, 56–57 evolution of sequences, 545–47 genetic information in, 22 history as genetic material, 76–77 hybridization of, 102, 590–92 isolation of, 568 measurement of concentration with UV light, 582–83 melting of, 100–101 melting temperature of, 591–92 methylation of, 120–21 purification of, 568–69 gel electrophoresis, 570–72 removal of unwanted RNA, 569–70 radioactive labeling of, 583 recognition by restriction endonucleases, 601 recombinant, see cloning regulatory region, 60 restriction and modification of, 600–601 restriction map of, 604–7 sequencing of, 555–59 supercoiling, 91–92 synthesis of, 574–80 complete genes, 580 coupling, 579 in eukaryotes, 130 phosphodiester bonding, 665 precursors, 109–10 priming and elongation, 665 primosome in, 114–16 DnaE protein, 321 DNA fingerprinting, 84 DNA glycolases, 383–84 DNA gyrase, 90, 106–7 DNA helicase, 107 DNA libraries, see gene libraries DNA ligase, 117, 603–4, 605 DNA microarrays, 709–12 DNA polymerase III (Pol III), 111–12 proofreading, 113 subunits of, 115 DNA polymerase I (Pol I), 117 DNA polymerases, 107–9 in chromosome replication, 130 for DNA sequencing, 668 elongation of DNA strands, 111–12 proofreading, 351 vs RNA polymerase, 542 source of, 33 spontaneous mutations, 351–52 DNA polymerase V, 389, 390 DNA repair systems, 378–79 double-strand repair, 392 of Escherichia coli, 379 in eukaryotes, 391–92 excision by specific bases, 383–84 gene conversion, 392–95 general excision repair, 381–83 mismatch repair, 379–81 photoreactivation, 386 recombination, 388 SOS error-prone repair, 388–91 specialized repair mechanisms, 384–86 transcription-coupled repair, 387–88 Dnase I (deoxyribonuclease I), 705–6 DNA sequencing, 663 automated, 670–71 bioinformatics, 690–92 chain termination method, 663–68 DNA chip technology, 672–73 DNA polymerases for, 668 exon trapping, 688–91 mapping of sequence tagged sites, 676–79 nanopore detectors, 676–77 oligonucleotide array detector, 672–74 in personal genomics, 689 primer walking, 670–71 pyrosequencing, 674–75 sequence polymorphism, 686–88 shotgun sequencing, 680–81 template DNA for, 668–70 DNA viruses, 49, 466–67 dnaX, 113 domains (of life), 38 domains (of protein), 166–67 dominant alleles, donor cells, 485 double helix, 57 double-strand repair, 392 downstream sequence elements (DSE), 332 Drosophila melanogaster, 18 haploid genome of, 42 Hox genes, 530–32 telomeres, 129 Drosophila virilis, 84 DSE (downstream sequence elements), 332 Ds elements, 411–12 Duchene muscular dystrophy, 628 duplications, 335 creation of new genes, 547–49 tandem, 344–45 E early genes, 457 Earth, formation of, 534 ECFP (enhanced cyan fluorescent protein), 699 Echinodermata, 40 EDTA (ethylene diamine tetraacetate), 568 eelworms, 41 E (exit) site, 213 effective genome, 358 EGFP (enhanced green fluorescent protein), 699 EJC (exon junction complex), 330 elastase, 180 electromagnetic radiation, 350 electron microscope, 588–90 electrophoresis, 570 electrospray ionization (ESI), 722–23, 725 elongation factors, 213 EMS (ethyl methane sulfonate), 348 enantiomers, 158 Encephalitozoom cuniculi, 78 endonucleases, 327, 600 endoplasmic reticulum, 35 endosymbiosis, 510–12 energy of activation, 182–84 enhanced cyan fluorescent protein (ECFP), 699 enhanced green fluorescent protein (EGFP), 699 enhanced yellow fluorescent protein (EYFP), 699 enhancers, 149, 152–53, 265–68 Entamoeba, 37–38 Index Enterococcus faecalis, 420 enterotoxins, 446 environmental analysis, 653–54 enzymes, 3–4 as catalysts of metabolic reactions, 177–79, 182 chemical modification of, 189–90 and energy of activation, 182–84 names of, 181 proteins as, 70, 174 rate of reactions, 184 regulation of, 187 specificities of, 179–80 substrate analogs, 184–85 substrate-binding models, 181 Epulopiscium fishelsoni, 27 error-prone repair, 388–91 erythromycin, 230 Escherichia coli, 17–20 2D protein gels of, 235 chromosome, 19 ColE1 plasmids of, 610 conjugating cells of, 498 CsrAB regulatory system of, 283 DNA polymerase errors in, 351 DNA repair systems of, 379, 388–91 DNA replication in, 120 genes, 663 heat shock response in, 238–39 K-12 strain of, 31 as model bacterium, 31–32 pathogenicity of, 30 plasmids, 20 size of, 27 two-component regulatory system in, 254 ESI (electrospray ionization), 722–23, 725 essential amino acids, 517 EST (expressed sequence tag), 676–79 ethidium bromide, 571–72 ethylene diamine tetraacetate (EDTA), 568 ethylene methane sulfonate (EMS), 348 eubacteria, 28 vs archaebacteria, 554–55 classification of, 38 gene transfer in, 504–5 euchromatin, 96, 684 eukaryotes, 26 cell cycle, 131 cell lineages, 36 chromosomes, 129–30 classification of, 38–40 defining features of, 509 diversity of, 36 DNA repair in, 391–92 genome of, 35, 87 genome sizes, 79 initiation complex, 220 multiple cell compartments in, 34–36 protein synthesis in, 218–21 replicating linear DNA in, 126–29 symbiotic theory of organelle origins, 37, 509–10 transcriptional regulation in, 263 DNA looping, 268 enhancer and insulator sequences, 265–68 imprinting, 275–77 mediator complex, 264–65 methylation of DNA in, 273–75 negative control, 269–70 silencing, 275 transcription factors, 264 transcription in, 145–46 protein-encoding genes, 148–51 ribosomal RNA (rRNA), 146–47 transfer RNA (tRNA), 146–47 yeasts, 40–41 evolutionary time scale, 535 excisionase, 375–76 excision repair system, 381–83 exit (E) site, 213 exon junction complex (EJC), 330 exons, 79 exon trapping, 688 procedure, 691 vector, 690 exonucleases, 126, 327, 600 expressed sequence tags (ESTs), 676–79 expression sites, 524 expression vectors, 631–33 exteins, 318–19 extinct animals, 562–64 extragenic suppression, 361 EYFP (enhanced yellow fluorescent protein), 699 F FACS (fluorescence activated cell sorter), 586 FadR protein, 249 families, 40 family trees, standard symbols for, 15 FCMD (Fukuyama-type congenital muscular dystrophy), 414 Fe4S4 cluster, 285–86 fermentation, 37 ferric uptake regulator (Fur), 290 ferritin, 175, 284–86 fertility plasmids, 496 filial generation, 12 FISH (fluorescence in situ hybridization), 595–97 FLAG® tag, 726 Flammulina velutipes, 430 Flp recombinase, 450–51 Flp recombination target (FRT), 451 fluorescence, 585–87 fluorescence activated cell sorter (FACS), 586 fluorescence in situ hybridization (FISH), 595–97 775 fluorescence resonance energy transfer (FRET), 655–56 fluorescent DNA sequencing, 670–71 fluorescent polymerase chain reaction, 655–56 fMet (N-formyl-methionine), 210 Fnr protein, 252 folate, 109 footprint analysis, 705–6 fosmidomycin, 514 F-plasmids, 31 formation of, 502 host-killing operon of, 435–36 insertion sequences, 499 integration into chromosomes, 496– 501 in transfer of chromosomal genes, 500 frameshift mutation, 226 production of abnormal proteins, 343–44 second-site reversion of, 360 Franklin, Rosalind, 77 FRD (fumarate reductase), 361 FRET (fluorescence resonance energy transfer), 655–56 FRT (Flp recombination target), 451 fruit flies, 18 haploid genome of, 42 Hox genes, 530–32 telomeres, 129 Fukuyama-type congenital muscular dystrophy (FCMD), 414 fumarate reductase (FRD), 361 functional genomics, 694 fungi, 39 Fur (ferric uptake regulator), 290 fusidic acid, 230–31 fusion tags, 726–29 G galactose, 696 galactosidase, 178 galactosides, 247 gametes, 7, 11–12 gametophytes, 45 gaps, 116–17 GC ratio, 101 gel electrophoresis, 570–72 denaturing gradient, 573–74 of protein, 719–20 pulsed field, 572–73 in separation of topoisomers, 92 gel retardation assay, 704 gene cassette, 606 GeneChip® array, 673 gene conversion, 392–95 in repair of broken chromosomes, 321–22 variant surface glycoprotein (VSG), 525–26 gene creatures, 49–50, 397 776 Index gene expression, 133–34 DNA microarrays for, 709–12 gene fusions, 699–702 monitoring, 694 assayable enzymes, 696 green fluorescent protein (GFP), 699 light emission by luciferase, 696–99 reporter genes, 694–96 prevention of, 293–94 primer extension, 706–7 serial analysis of, 713–16 transcriptome analysis, 709 upstream region, 702–5 gene fusions, 699–702 gene libraries, 621–23 creating, 622 screening by hybridization, 623 screening by immunological procedures, 623–24 screening by probing, 624 genera, 40 generalized transduction, 493–94 generation time, 124 gene regulation, 235–36 activators, 243–44 efficiency vs rapid response in, 237 negative control, 143, 236, 243–44, 269–70 operon model of, 244–46 phosphorelay systems, 254 positive control, 143, 236, 243–44 repressors, 243–44 specific vs global control, 254–55 at transcription level, 236–37 two-component regulatory system, 253–54 genes, arrangement in chromosomes, chemical synthesis of, 580 duplication of, 547–49 etymology of, evolution of sequences, 545–47 families of, 548 genetic information in, 22 inserting into vectors, 610–12 modifier, 11 orthologous, 549–50 paralogous, 549–50 superfamilies of, 548 gene superfamily, 547 genetic code, 66 and amino acid sequence of proteins, 67–69 decoding, 199–200 and ribosomes, 65–67 universal, 227 genetic elements, 397 Gengis Khan, 563 genome, contigs, 683 effective, 358 eukaryotic, 87 genetic information in, 22 shotgun sequencing of, 683 sizes of, 79 genome mining, 690–92 genomics, 663 functional, 694 personal, 689 genotypes, genus, 38 germline cells, 36, 45 GFP (green fluorescent protein), see green fluorescent protein Giardia, 37 Gilbert, Walter, 541 global regulation, 245, 254–55 globin, 547–48 GloFishTM, 43 glutamine tRNA, 206 glutathione-S-transferase (GST), 726 glycine, 155 glycogen, 190, 191 glycoproteins, 172–73 glycosidases, 182 Golgi apparatus, 35 Gosling, Raymond, 77 G-phases, 131 gram-negative bacteria, 32, 501–3 gram-positive bacteria, 32, 501–4 gratuitous inducer, 246 green fluorescent protein (GFP), 699 for protein localization, 701 source of, 177 in transgenic organisms, 700 GST (glutathione-S-transferase), 726 GTP (guanosine triphosphate), 308 guanidine, 196 guanidinium chloride, 196 guanine, 54–56, 60, 385 guanosine triphosphate (GTP), 308 guide RNAs, 69, 326 GyrA protein, 90 gyrase, 90 H Haemophilus influenzae, 680 hairpin, 87 halobacteria, 555 haploid genome, haploidy, 7, 29, 45, 277 HAT (histone acetyl transferase), 271 Hazara tribe, 563 HDAC (histone deacetylase), 271 H-DNA, 91, 94–95 HDV (hepatitis delta virus), 480 headful packaging, 494 heat shock protein (HSP), 224–25, 238–39 heat shock response, 238–40 heat-unstable (HU) nucleoid protein, 257–58 helicase, 107 helix-loop-helix (HLH) motif, 191–93 helix-turn-helix (HTH) motif, 191–93 helper phages, 495 helper viruses, 479 heme, 170 hemi-methylated DNA, 381 hemoglobin, 168, 547 hemolysin, 446, 676 Hemophilus influenzae, 506 hepatitis delta virus (HDV), 480 heredity, 59–60 herpes viruses, 466–67 heterochromatin, 96 access to DNA, 270–73 in coding DNA, 236 in satellite DNA, 84 heterozygous, Hfr-strains, 499 higher organisms, 27; see also eukaryotes cell division in, 130–31 recombination in, 376–78 repetitive DNA sequences in, 81–83 transposons in, 410–12 histidine, 228 histone acetyl transferase (HAT), 271 histone deacetylase (HDAC), 271 histone-like proteins, 256–57 histones, 95–98 HIV (human immunodeficiency virus), 477 HLH (helix-loop-helix) motif, 191–93 Holliday junction, 370–71 migration of, 373 rearrangement and resolution of, 372 homeobox genes, 530–32 homeodomain, 530–32 homing introns, 423–24 Homo erectus, 560 homologous chromosomes, 7–8 homologous recombination, 369–70, 486–87 Homo sapiens, 560 homozygous, horizontal gene transfer, 507, 564–66 hot spots, 355–58 hot springs, 28 housekeeping genes, 134 CG-islands in, 274 constitutive expression of, 140 Hox genes, 530–32 HSP (heat shock protein), 224–25, 238–39 HTH (helix-turn-helix) motif, 191–93 HU (heat-unstable) nucleoid protein, 257–58 human chromosomes, 63–64 human genome, 44 shotgun sequencing of, 683 survey of, 683–86 Human Genome Project, 680–82 human immunodeficiency virus (HIV), 477 human mitochondrial DNA, 512 humans, 44 hybrid DNA, 102, 590–92 Index hydrogen bonds, 57, 160–63, 165 hydrolase, 182 hydrophilic amino acids, 156–58 hydrophobic amino acids, 156–58 hypoxanthine, 202 I IBP (insulator binding protein), 265 ice nucleation factors, 174 IHF (integration host factor), 257–58 imino acid, 158 imitation switch (ISWI) complexes, 272 immunity proteins, 445–46 immunization, 48 immunological screening, 623–24 imprinting, 275–77 induced fit model, 181 induced mutations, 348 inducers, 143, 243 infectious diseases, 34 informational macromolecules, 540 information processing proteins, 177 inheritance, 15–16, 511 inherited diseases, 334–35 initiation complexes, 211 assembly of, 220 components of, 118 initiation factors, 211, 222 initiator box, 149 initiator tRNA, 210 inosine, 93, 202 insertional inactivation, 612 insertion mutations, 335, 341–43 insertion sequences, 400–401, 499 insulator binding protein (IBP), 265 insulators, 265–68 insulin, 552 integrase, 374 integration host factor (IHF), 257–58 integrons, 420–22 inteins, 318–21, 729–30 intercalation, 348 intergenic DNA, 79 intergenic regions, 60–62 internal eliminated segments (ISEs), 518–20 internal resolution site (IRS), 402 internal ribosome entry sites (IRES), 219 interphase, 131 intervening sequences, 79–80 Int protein, 374 intracellular parasites, 455 intragenic suppression, 361 introns, 79–80 classes of, 310 DNA sequences of, 552 homing, 423–24 removal from RNA, 310–14 self-splicing, 541–42 splicing mechanism, 314–15 inverse polymerase chain reaction (PCR), 641–42 inversions, 335 DNA rearrangement with, 343–45 and phase variation, 346 invertase, 345–46 inverted repeats, 86, 398 in vitro mutagenesis, 365–66, 367 in vivo mutagenesis, 365–66 ionizing radiation, 350 IPTG (iso-propyl-thiogalactoside), 246 IRE (iron-responsive element), 285–86 IRES (internal ribosome entry sites), 219 iron regulatory protein (IRP), 285–86 iron-responsive element (IRE), 285–86 iron sulfur cluster, 252 IRP (iron regulatory protein), 285–86 irreversible inhibition, 185 IRS (internal resolution site), 402 ISEs (internal eliminated segments), 518–20 isolectric focusing, 720 isomerases, 182 iso-propyl-thiogalactoside (IPTG), 246 isoschizomers, 602 ISWI (imitation switch) complexes, 272 J J-base, 524 Jenner, Edward, 468 jumping genes, 397 junk DNA, 84–85, 422–23 K kappa particles, 515–16 karyotype, 63 killer Paramecium, 515–17 kilobase ladder, 572 kinases, 109, 182 kinetic proofreading, 111–12 kinetochores, 62 kinetoplasts, 522 kingdoms, 38–40 Klenow polymerase, 668 Kluyveromyces lactis, 430 knotted DNA, 91 L LacI protein, 143, 245–46 lac operator, 145, 256 lactamase, 173, 182 lactose, 247–48 lactose permease, 245 lacZ gene, 696 lagging strand, 108, 116–18 lambda attachment sites, 494 lambda DNA, integration of, 376–77 lambda left promoter, 633 lambda replacement vectors, 616–18 lambda repressor, 633 lambda viruses, 460 lariat structure, 312 late genes, 457 lateral gene transfer, 507, 564–66 777 LDL (low density lipoprotein), 324, 550 leader peptidase, 223 leader peptide, 298 leader region, 297 leading strand, 108 leaky mutation, 335 Lederberg, Joshua, 31 Leishmania, 572 leucine zipper, 192 LexA protein, 389 life, 22–23 ligase, 128, 182 LINE-1 element, 415 linear plasmids, 428–30 LINE (long interspersed element), 82, 414–15 linkage, 16 linkage groups, 17 linking number (L), 89 lipoproteins, 172–73 living cells, 454–55 lock and key model, 181 locus, 78 long interspersed element (LINE), 82, 414–15 long terminal repeats (LTRs), 413, 475 low density lipoprotein (LDL), 324, 550 lower organisms, 27 LTRs (long terminal repeats), 413, 475 luciferase, 177, 696–99 luciferin, 696–99 lumi-phos, 588 Lyme disease, 126 lysine, 229 lysogen, 451–52, 460 lysogeny, 451–52, 460–62 lysosomes, 35, 232 lysozyme, 166, 568 lytic growth, 451–52, 460 M macromolecules, 22 macronucleus, 518–19 macronucleus-destined segment (MDS), 518–20 Magnetobacterium bavaricum, 176 magnetosomes, 176 maintenance methylases, 274 malaria, 512–15 MALDI (matrix-assisted laser desorption-ionization), 722–23, 725 malE gene, 728 maltose, 142–43 maltose-binding protein (MBP), 726–29 MalT protein, 142 mammals, repetitive DNA of, 414–15 map units, 19 mariner transposon, 412 MAR (matrix attachment region), 268 mass spectrometry, 722–26 mating factors, 528 mating types, 527 778 Index MAT locus, 527–30 matrix-assisted laser desorptionionization (MALDI), 722–23, 725 matrix attachment region (MAR), 268 maximum velocity, 184 MBP (maltose-binding protein), 726–29 McClintock, Barbara, 410 MCS (multiple cloning site), 611–12, 729 MDS (macronucleus-destined segment), 518–20 mechanical proteins, 175–76 MeCP (methylcytosine binding protein), 275 mediators, 264–65 megabase chromosomes, 524 meiosis, 12, 16–17 melting temperature, 100 membrane, 25 membrane-bound organelles, 35 Mendel, Gregor, Mendelian characters, Mendelian ratios, 12 messenger RNA (mRNA), 133; see also gene expression; RNA attachment of ribosomes to, 214 capping of, 306–8 in chromosomes of higher organisms, 63 cleavage of, 283, 285 degradation of, 282–84, 327–28 elongation of, 137 eukaryotic, 305–6 functions of, 25 hybridization of, 710 monocistronic vs polycistronic, 135 nonsense mediated decay of, 328–32 poly(A) tail, 308–10, 311 polycistronic, 215–16 purification by oligo(dT), 626 RNA editing of, 324–26 termination of, 140 metabolism, 22, 544–45 metabolites, 365 metabolomics, 741–44 metallothionein, 175 Methanobacterium thermoautotrophicum, 506 methanogens, 555 methotrexate, 109 methylases, 182 methylated bases, 380 methylation, 120–21 in control of gene expression, 273–75 in genetic imprinting, 275–77 in silencing of genes, 275 methylcytosine binding protein (MeCP), 275 mice, 44 Michaelis constant, 184 Michaelis-Menten equation, 184 micronucleus, 518–19 micro RNA (miRNA), 295–97 Miescher, Frederich, 76 Miller, Stanley, 536 Miller experiment, 536–38 mini-chromosomes, 522–24 mini-satellites, 84 miRNA (micro RNA), 295–97 mirror-like palindromes, 86 mismatches, 111–12 mismatch repair system, 379–81 functions of, 112 MutSHL, 382 mispairing, 353 of direct repeats, 354 of inverted repeats, 355 missense mutations, 336–38 mistranslation, 226 mitochondria, 35 genome of, 510–11 in protein synthesis, 225–26 mitochondrial DNA, 512, 559–60 mitosis, 131 mitosomes, 37 mobile DNA, 397; see also DNA homing introns, 423–24 junk DNA, 422–23 repetitive DNA, 414–15 selfish DNA, 422–23 transposable elements, 397–98 mobile genetic elements, 341 mobility shift assay, 703 moderately repetitive sequences, 82 modification enzymes, 600–601 modified bases, 201–2, 203 modifier genes, 11 molecular beacons, 598, 656 molecular chaperones, 224–25 molecular cloning, see cloning molecular evolution African Eve hypothesis, 559–61 ancient DNA, 562–64 archaebacteria vs eubacteria, 554–55 autotrophic theory, 544–45 DNA sequencing, 555–59 early atmosphere, 534–35 evolution of gene sequences, 545–47 first cells, 542–44 formation of Earth, 534 gene duplication, 547–49 horizontal gene transfer, 564–66 informational macromolecules, 540 Miller experiment, 536–38 mitochondrial DNA, 559–60 Oparin’s theory of the origin of life, 535–36 paralogous and orthologous genes, 549–50 polymerization of monomers, 538–39 proteinoids, 539–40 rates of evolutions for proteins, 550–51 ribosomal RNA (rRNA), 552–54 ribozymes, 540–42 molecular sewing, 649 Mollusca, 39–40 Mongols, 563 monocistronic mRNA, 135 monomers, polymerization of, 538–39 mRNA, see messenger RNA Mullis, Kary, 637 multi-cellular organisms, 530–32 multimeric proteins, 167 multiple cloning site (MCS), 611–12, 729 multiregional model of evolution, 561 mutagenic chemicals, 348–50, 363–64 mutations, 334 base substitution, 336 biochemical pathway of, 4–5 causes of, 348–53 chemical mutagens, 348–50 DNA polymerase errors, 351–52 mispairing, 353 radiation, 350–51 recombination, 353 conditional, 338 deletion, 340–41 experimental isolation of, 364–65 frameshift, 343–44 hot spots, 355–58 insertion, 341–43 missense, 336–38 nonsense, 338–40 phase variation, 345–46 rates of, 358–59 reversions, 359–61 silent, 346–48 site-directed mutagenesis, 366–67 spontaneous, 348 by chemical instability, 353–55 by DNA polymerase errors, 351–52 by tautomerization, 353 temperature-sensitive, 338–39 types of DNA sequence alteration, 335 in vivo vs in vitro mutagenesis, 365–66 mutator genes, 351 MutSHL mismatch repair system, 382 mutualism, 515 Mycobacterium, 653 Mycoplasma genitalium, 78 MyoD transcription factor, 269–70 Myxococcus, 78 N NAD (nicotinamide adenine dinucleotide), 178–79 nalidixic acid, 90 Nanoarchaeum equitans, 78 nanopore detectors, 676 nanotechnology, 177 natural competence, 492 Neanderthal Man, 561–62 negative feedback, 187 negative regulation, 236; see also gene regulation in eukaryotes, 269–70 by repressor proteins, 143, 243–44 Index negative supercoiling, 88–89 Nematoda, 39–40 nematodes, 41–42 neomycin phosphotransferase, 441 Neurospora, 294–95 New England Biolabs, 731 N-formyl-methionine (fMet), 210 nicks, 117 nick translation, 118 nicotinamide adenine dinucleotide (NAD), 178–79 NMD (nonsense mediated decay), 328– 32 Noah’s Ark model of evolution, 560– 61 non-coding DNA, 41, 62, 78–80 non-coding RNA, 283, 304 non-coding strands, see template strands non-homologous end joining, 392, 393 non-homologous recombination, 369 nonsense mediated decay (NMD), 328– 32 nonsense mutations, 338–40, 362–63 nonsense suppression, 362 non-template strands, 133 norfloxacin, 90 Northern blotting, 594 novobiocin, 90 N-terminus, 71, 155 nuclear envelope, 34–35 nuclear matrix, 268 nuclear pores, 35 nucleases, 182, 600 nucleic acids, 52 absorption of UV radiation by, 582 bases, 54–56 blotting, 592–95 chemical structure of, 52–54 chemical tagging of, 587–88 degradation of, 600 detection of, 583–85 autoradiography, 584–85 scintillation counting, 583–84 genetic information in, 22 hybridization of, 590–92 primeval synthesis of, 540 radioactive labeling of, 583 nucleocapsid, 457 nucleolar organizers, 146 nucleomorph, 511 nucleoproteins, 172 nucleosides, 55 nucleotides, 52 components of, 55 ordering of, 53 phosphodiester linkages, 54 polymerization of, 109–10 structure of, 53 nucleus, 26 null alleles, NusA protein, 259–61 O oil drop model, 164–65 Okazaki fragments, 113 joining, 117 primer for, 116 oligo(dT), 626 oligonucleotide array detector, 672–74 oligonucleotide arrays, 710–12 ONPG (ortho-nitrophenyl galactoside), 185, 696 opal, 363 Oparin, Alexander, 76, 535–36 open circle, 89 open reading frame (ORF), 134–35 in insertion sequences, 400 and nucleotide sequences, 209 operators, 143 operons, 62 in gene regulation, 244–46 transcription of, 135, 215–16 optical isomers, 158 orders, 40 ORF, see open reading frame organelles, 35 origin of chromosome (oriC), 19, 118 origin of replication, 118 orthologous sequence, 549–50 ortho-nitrophenyl galactoside (ONPG), 185, 696 overlap primer, 649 oxidoreductases, 182 OxyR protein, 252 Oxytricha, 520, 521 P P1 artificial chromosome (PAC), 621 P1 virus, 451–52, 494 P22 virus, 494 PABP (poly(A)-binding protein), 308–10 PAC (P1 artificial chromosome), 621 PAGE (polyacrylamide gel electrophoresis), 720–22 palindromes, 86 paralogous sequence, 549–50 Paramecium, 515–17 DNA sequences in, 128 macronucleus of, 519 micronucleus of, 519 parasites, 46, 455 parasitism, 515 partial diploids, 20 partial dominance, patch recombinants, 371 pathogenic bacteria, 30 pathogenicity islands, 506–7 pathogens, 515 PCNA protein, 130 PCR, see polymerase chain reaction PCR (inverse polymerase chain reaction), 641–42 peas, 2–3 penetrance, 10–11 779 penicillins, 33, 439 peptide bonds, 70–71, 155 peptide nucleic acid (PNA), 580–82 peptide (P) site, 213 peptidoglycans, 158–60, 568 peptidyl transferase, 208 Perlegen Sciences, 689 permeases, 174 personal genomics, 689 pesticins, 443 Petri dishes, 30–31 PFGE (pulsed field gel electrophoresis), 572–73 phages, see bacteriophages pharmacogenomics, 688 phase variation, 345–46 phenol extraction, 568–69 phenotypes, pheromones, 491, 504, 528 phoA gene, 696 phosphatases, 182, 190 phosphate group, 52, 584 phosphodiester, 52, 54 phospholipids, 23, 25 phosphoramidites, 575–76 phosphorelay systems, 254 phosphorothioate, 584 phosphorylation, 190 phosphoramidates, 538–39 photolyase, 386 photoreactivation, 386 photosynthesis, 544 phyla, 39–40 planets, formation of, 534 plants, 39 plant viruses, 466–67, 470–71 plasmids, 20 2-micron, 450–51 addiction, 435–36 aggressive characters from, 442–44 antibiotic resistance, 436–38 aminoglycosides, 440–41 beta-lactam antibiotics, 438–39 chloramphenicol, 439–40 sulfonamides, 442 tetracyclines, 441–42 trimethoprim, 442 benefits to host cells, 436 copy number, 432–35 F-plasmids, 31, 435–36, 496–502 functions of, 49 host killing functions, 435–36 incompatibility of, 428, 429 integration of, 496–501 linear, 428–30 lysogeny, 451–52 properties of, 427–28, 437 replication of, 430–32 as replicons, 426–27 ribonucleic acid (RNA), 430 structure of, 126 Ti-plasmids, 447–50 780 Index transferability of, 428, 495–96 virulence, 446–47 Plasmodium, 513–15 plastids, 510 Platyhelminthes, 39–40 ploidy, pMAL vectors, 728 PNA (peptide nucleic acid), 580–82 Pneumococcus pneumoniae, 488 point mutation, 335 Pol I (DNA polymerase I), 117 Pol III (DNA polymerase III), 111–12 proofreading, 113 subunits of, 115 poly(A)-binding protein (PABP), 308–10 polyacrylamide, 570–71 polyacrylamide gel electrophoresis (PAGE), 720–22 polyadenylation complex, 308 poly(A) polymerase, 308–10 poly(A) tail, 308–10, 311 polycistronic mRNA, 135 in bacteria, 215–16 operons, 244 polydactyly, 11 polyhistidine tag (His tag), 726–27 polylinker, 611–12 polymerase chain reaction (PCR), 635–36 artificial restriction sites, 642–43 cycles, 638–40 degenerate primers, 640–41 differential display, 647–48 directed mutagenesis, 651 engineering deletions and insertions by, 651–52 in environmental analysis, 653–54 fluorescent, 655–56 in genetic engineering, 649 invention of, 637 inverse, 641–42 in medical diagnosis, 652–53 primers, 635 randomly amplified polymorphic DNA (RAPD), 643–46 rapid amplification of cDNA ends (RACE), 649, 650 in rescuing DNA from extinct life forms, 654–55 reverse transcriptase, 646–47 rolling circle amplification technology (RCAT), 657–61 scorpion primers, 656–57 TA cloning by, 643 polymerase Eta, 391 polymerases, 107, 182 polymerization, 538–39 polymorphism, 686–88 polypeptides, 65; see also proteins components of, 70, 155–58 formation of, 155 structures of, 160 polyphosphates, 538 polyploidy, 45 polyproteins, 199, 469–71 polysomes, 214, 216 Porifera, 39–40 positive regulation, 143, 236, 243–44 post-transcriptional gene silencing (PTGS), 294–95 post-translational modifications, 227 poxviruses, 466–67 P (peptide) site, 213 precursors, synthesis of, 110 PriA protein, 114 Pribnow box, 136 primary atmosphere, 534 primary endosymbiosis, 511–13 primary structure, 71–72, 160 primary transcript, 80 eukaryotic vs prokaryotic mRNA, 283 in RNA processing, 303 transcription of protein-encoding genes, 149 primase, 108–9, 114 primer extension, 706–7 primer walking, 670–71 primitive soup, 535–36 primosome, 114 prions, 50, 481–83 probe molecules, 592 processed pseudogene, 415 prokaryotes, 26 genome sizes, 79 protein synthesis in, 218–19 proline, 185 promoters, 135, 149 proofreading, 351 prophages, 376, 460, 494 prosthetic groups, 71, 160 proteases, 231–33 active site specificity of, 180 in degradation of heat-damaged proteins, 239 functions of, 182 proteasomes, 232, 233 protein A, 726 Protein Data Bank, 171 protein-encoding genes, 148–51 protein kinases, 190 protein machines, 177 proteinoids, 538 enzyme activities of, 539–40 formation of, 539 protein primers, 127 proteins, 155 biological roles of, 73–74 cellular functions, 70–71, 174–77 cotranslational export of, 223 degradation of, 231–33 denaturation of, 194–96, 719 DNA-binding, 190–94, 702–5 domains of, 166–67 evolution of sequences, 545–47 export of, 221–23 exteins, 319 gel electrophoresis of, 719–20 as gene products, 198–99 in gene regulation, 246–48 genetic code, 67–68 inteins, 319 interactome analysis of, 732–37 in living cells, 23 mass spectrometery of, 722–26 microarrays, 741–43 molecular weight of, 705 multimeric, 167 primeval synthesis of, 540 rates of evolution, 550–52 structure of, 71–73, 160 3-D, 165–66 quaternary, 167–68 secondary, 160–63 tertiary, 163–64 synthesis of, 65 in chloroplasts, 225–26 discontinuation of, 221 eukaryotic vs prokaryotic, 218–21 genetic information in, 198 inhibition by antibiotics, 230–31 initiation complexes, 210 mistranslation, 226 in mitochondria, 225–26 reading frames, 208–9 start codon, 209–10 termination, 213–14 transfer RNA (tRNA) sites, 210–13 tagging of, 726 fusion tags, 726–29 inteins, 729 two-dimensional PAGE of, 720–22 Western blotting of, 722, 724 proteolipids, 173–74 proteome, 718 proteomes, 199 proteomics, 718–19 co-immunoprecipitation, 737–40 gel electrophoresis, 719–22 mass spectrometry, 722–26 metabolomics, 741–44 phage display, 729–32 protein arrays, 741–43 protein tagging systems, 726–29 tethering technology, 734 Western blotting, 722 yeast two-hybrid system proto-cells, 542–43 protoctista, 38–40 protomers, 167 protostomes, 531–32 provirus, 460 pseudogenes, 81–82, 415–16 Pseudomonas aeruginosa, 242 pseudouridine, 323 pSPL vector, 690 PTGS (post-transcriptional gene silencing), 294–95 Index PtsG protein, 251 pulsed field gel electrophoresis (PFGE), 572–73 Punnett squares, 13 purine, 54–56 pyrimidine, 54–56, 388 pyrosequencing, 674–75 pyrrolysine, 228–30 Q quaternary structure, 71–72, 160, 167–68 quenchers, 657 quinolone, 90, 107 R racemases, 158, 182 RACE (rapid amplification of cDNA ends), 649, 650 radiation, 350–51 radiation hybrid, 679 radical replacement, 337–38 Rad proteins, 378 RAPD (randomly amplified polymorphic DNA), 643–46 rapid amplification of cDNA ends (RACE), 649, 650 R-bodies, 516–17 RBS (ribosome binding site), 210 RCAT (rolling circle amplification technology), 657–61 RdRP (RNA-dependent RNA polymerase), 292 reading frames, 208–9 RecA protein, 373, 375, 389 RecBCD protein, 374 recessive alleles, recipient cells, 485 recognition RNA, 69 recombinant DNA, see cloning recombination, 369 chi sites, 373 in DNA repair, 388, 389 in higher organisms, 376–78 homologous, 369–70, 486–87 during meiosis, 16–17 mutations from, 353 non-homologous, 369 single-strand invasion, 371–73 site-specific, 373–76 timeline of, 377 topoisomerase IV, 123 regulatory proteins, 73–74; see also proteins defects in, as repressors and activators, 246–48 in translational activation, 287–88 in translational repression, 284–86 regulatory region, 60–62 regulon, 255 release factor (RF), 213–14, 215 renaturation, 102 repeated sequences, 81 repetitive DNA, 414–15 repetitive sequences, 81 replica plating, 366 replication, 63 functions of, 22 generation time, 124–25 of plasmid DNA, 430–32 proteins involved in, 120 rolling circle, 430–33, 498 semi-conservative, 105, 130 stages of, 104–5 and supercoiling, 105–7 termination of, 121–22 replication bubbles, 129 replication factor C (RFC), 130 replication fork, 105, 112–14 replication licensing factors (RLF), 130 replicative form (RF), 462, 668 replicative transposition, 402–6, 407 replicons, 125–26, 398, 426–27 replisomes, 105 reporter genes, 694–96; see also gene expression in antibiotic resistance, 695 assayable enzymes, 696 gene fusions, 699–701 green fluorescent protein (GFP), 699 luciferase, 696–99 reporter proteins, 177 repressors, 143–44 functions of, 243–44 modification of, 252 regulatory proteins as, 246–48 resolvase, 371, 402 restriction endonucleases, 601–2 restriction enzymes, 600; see also enzymes cutting of DNA by, 602–3 naming of, 601–2 recognition sequence, 602 source organisms, 602 type I, 602–3 type II, 603 restriction fragment length polymorphism (RFLP), 607–8, 609 restriction map, 604–7 retro-elements, 412–14 retrons, 416–17 retroposons, 341, 412–14 retro-pseudogene, 415 retroviruses, 472–77; see also viruses DNA integration and transcription in, 476 gene products and proteins, 478 genome of, 49, 477–78 in human genome, 479 ssRNA molecules in, 476 structure of, 474 reverse transcriptase, 412, 472, 475 reverse transcriptase PCR (RT-PCR), 646–47 781 reverse transcription, 474 reverse turn, 162–63 reversions, 359–61 by compensatory changes in other genes, 361 detection of chemical mutagens by, 363–64 RFC (replication factor C), 130 RFLP (restriction fragment length polymorphism), 607–8, 609 RF (replicative form), 462, 668 RF (release factor), 213–14, 215 R-group, 70 rhabdoviruses, 464 Rhizobium, 515 Rho-dependent terminators, 139 Rho-independent terminators, 139 Rho protein, 139–40 ribonuclease H (RNase H), 117, 433 ribonuclease III, 283 ribonuclease P, 305, 541 ribonucleases, 327, 569–70, 600 ribonucleic acid, see RNA ribonucleosides, 55 ribonucleotide reductase, 109 ribonucleotides, 55 ribose, 52, 54, 666 ribosomal RNA (rRNA), 67 base modification of, 322–23 cleavage of, 305 coding sequences of, 552–54 functions of, 69 instant evolution of, 556–57 processing of, 305 secondary structure of, 208 transcription of, 146–47 ribosome binding site (RBS), 210 ribosome modulation factor (RMF), 221 ribosome recycling factor (RRF), 214 ribosomes, 65–67 3-D structure of, 207 alterations to, 290–91 attachment to messenger RNA, 214 in decoding cells, 204–8 in protein synthesis, 22, 26, 198 rescue of, 217–18 structural components of, 66 riboswitches, 299–300, 542 ribozymes, 69, 208, 480, 541 rickettsias, 47 right-handed helix, 57 RISC (RNA-induced silencing complex), 291 RLF (replication licensing factors), 130 R-loop analysis, 590 RMF (ribosome modulation factor), 221 RNA-dependent RNA polymerase (RdRP), 292 RNA editing, 324–26 RNAi, see RNA interference 782 Index RNA-induced silencing complex (RISC), 291 RNA interference (RNAi), 291–92; see also gene expression amplification and spread of, 292–93 experimental induction of, 295 in laboratory research, 293–94 RNA plasmids, 430 RNA polymerase I, 146 RNA polymerase II, 151–52 RNA polymerase III, 146 RNA polymerases, 133 artificial evolution of, 543 vs DNA polymerases, 108 in gene transcription, 146 internal promoter for, 148 RNA-dependent, 292 structure of, 138 T7, 632 in termination sequence, 138–40 in transcription bubble, 138 RNA primer, 108–9 RNA replicase, 464 RNA (ribonucleic acid), 52; see also DNA; nucleic acids bases, 54–56 blotting, 592–95 chemical tagging of, 587–88 classes of, 69–70 coding, 304 detection of, 584–87 autoradiography, 584–85 fluorescence, 585–87 evolution of sequences, 545–47 genetic information in, 22 hybridization of, 590–92 measurement of concentration with UV light, 582–83 non-coding, 304 processing of, 302–3 radioactive labeling of, 583 removal from DNA samples, 569–70 termination sequence, 139 three-hybrid system, 739 transport of, 327 in viruses, RNase H (ribonuclease H), 117, 433 RNA viruses, 467–69 bacterial, 469 double-stranded, 469 genome of, 49 negative-stranded, 470, 472 plant viruses, 470–71 positive-stranded, 469–70, 471 RNA world, 541 rolling circle amplification technology (RCAT), 657–61 rolling circle replication, 430–33, 498 rotaviruses, 470 roundworms, 41–42 R-plasmids, 436–38 RpoE protein, 238–39 RpoH protein, 238–39 RRF (ribosome recycling factor), 214 rRNA, see ribosomal RNA RT-PCR (reverse transcriptase PCR), 646–47 S S1 nuclease, 707–9 Saccharomyces cerevisiae, 40–41 2-micron plasmids of, 450–51 haploid and diploid phases of, 527 mating types, 525–30 SAGE (serial analysis of gene expression), 713–16 Salmonella, 232, 346, 506–7 Salmonella typhi, 447 Salmonella typhimurium, 363–64 sarin, 185 SAR (scaffold attachment region), 268 satellite DNA, 83–84 satellite RNA, 479 satellite tobacco necrosis virus (STNV), 479–80 satellite viruses, 480 scaffold attachment region (SAR), 268 Schistosoma japonicum, 726 scintillants, 583 scintillation counting, 583–84 scorpion primers, 656–57 scrapie, 481–82 scRNA (small cytoplasmic RNA), 304 SDH (succinate dehydrogenase), 361 SDS (sodium dodecyl sulfate), 195, 719–20 Sec, see selenocysteine SECIS (selenocysteine insertion sequence), 228 secondary atmosphere, 534 secondary endosymbiosis, 511–13 secondary structure, 71–72, 160–63 second-site revertants, 359–61 selenocysteine insertion sequence (SECIS), 228 selenocysteine (Sec), 227–28 delivery of transfer RNA (tRNA) with, 229 structure of, 229 self-assembly, 169, 471 selfish DNA, 85–86, 422–23 self-splicing, 314 self-splicing introns, 541–42 semi-conservative replication, 105, 130 sense RNA, 288 sense strands, 133 sensor kinase, 253 septum, 124 SeqA (sequestration) protein, 121 Sequenase, 668 sequence polymorphisms, 686–88 sequence tagged sites, 676–79 sequestration (SeqA) protein, 121 serial analysis of gene expression (SAGE), 713–16 serine, 185 sex chromosomes, 13–15 sex determination, 13–15 sex-linked genes, 14–15 sex pilus, 496 sexual reproduction, 485 Shigella, 436–38 Shine-Dalgarno sequence, 210–11 short interfering RNA (siRNA), 291, 293–94 short interspersed elements (SINEs), 82–83, 414 shotgun sequencing, 680–81, 683 shuttle vectors, 615–16 sigma subunit, 136 signal molecules, 144–45 functions of, 243 nature of, 248–52 signal sequence, 222 silencing, 257, 275 silent mutations, 346–48 simian virus 40 (SV40), 466 simple sequence length polymorphism (SSLP), 686–88 SINE (short interspersed element), 82–83, 414 single nucleotide polymorphism (SNP), 686–88 single-strand binding (SSB) protein, 107 single-strand invasion, 371–73 siRNA (short interfering RNA), 291, 293–94 site-directed mutagenesis, 366–67 site-specific recombination, 373–76 small cytoplasmic RNA (scRNA), 304 small nuclear ribonucleoprotein (snRNP), 310 small nuclear RNA (snRNA), 69, 304 small nucleolar RNA (snoRNA), 304, 322–23 Smart Cycler® TD, 658 snoRNA (small nucleolar RNA), 304, 322–23 SNP (single nucleotide polymorphism), 686–88 snRNA (small nuclear RNA), 69, 304, 310 sodium dodecyl sulfate (SDS), 195, 719–20 soil bacteria, 438 somatic cells, 12, 36, 45 SOS error-prone repair system, 388– 91 Southern blotting, 594 South-Western blotting, 595 specialized transduction, 494–95 specific regulation, 244–45, 254–55 S-phase, 131 spliceosome, 310–13 Index splicing, 310–12; see also introns alternative, 315–18 different mechanisms, 314–15 inteins, 318–21 spontaneous mutations, 348; see also mutations by chemical instability, 353–55 by DNA polymerase errors, 351–52 by tautomerization, 353 spores, 239–42 SSB (single-strand binding) protein, 107 SSLP (simple sequence length polymorphism), 686–88 ssrA tag, 218 Staphylococcus, 676–77 Staphylococcus aureus, 27 start codon, 209–11 stem and loop motif, 87 stem cells, 279 steroid receptors, 194 sticky ends, 603, 604 STNV (satellite tobacco necrosis virus), 479–80 stop codons, 199, 361 strawberries, metabolome analysis of, 744 Strep tag, 726 streptavidin, 713, 726 Streptococcus pneumoniae, 488–89 Streptomyces, 33, 429, 441 Streptomyces lividans, 126 streptomycin, 33, 230 structural genes, 134 structural proteins, 73, 174 substrate, 73 subtractive hybridization, 628–31 subviral agents, 477, 479 succinate dehydrogenase (SDH), 361 suicide demethylase, 386 sulfhydryl group, 158 Sulfolobus, 225, 555 Sulfolobus solfataricus, 506 sulfonamides, 109, 442 supercoiling, 88–89 effect on DNA structure, 91–92 local, 91 and replication, 105–7 suppressor mutation, 359, 362–63 suppressor tRNAs, 362–63 SV40 (simian virus 40), 466 S-values, 65–67 Swi/Snf complexes, 272 SYBR® Green I probe, 655–56 symbiosis, 510, 515 symbiotic theory, 510 synthase, 182 T T4 ligase, 604 T7 RNA polymerase, 632 TA cloning, 643 tail specific protease, 218 tandem duplication, 344–45 tandem mass spectrometry, 726 tandem repeats, 83 TaqMan® probe, 655–56 Taq polymerase, 636 target DNA, 623 target sequence, 398 TATA binding protein (TBP), 149 TATA box, 149 Tatum, E.L., tautomerization, 353, 356 TBP (TATA binding protein), 149 Tc1 element, 411–12 T-DNA (tumor-DNA), 448–50 telomerase, 62, 128 telomeres, 62, 127 temperature-sensitive mutation, 338–39 template strands, 104–5 and sequence of RNA message, 133 slippage in, 352 teratogen, 349–50 terminators, 138–40 terminus of replication (ter), 19, 121–22 tertiary atmosphere, 534–35 tertiary structure, 71–72, 160, 163–64 tethering technology, 734 tetracyclines, 230, 441–42 tetrahydrofolate (THF), 109, 442 Tetrahymena, 128, 314 tetraploidy, thermocyclers, 636 Thermotoga, 507 Thermus aquaticus, 33, 636 theta-replication, 106 THF (tetrahydrofolate), 109, 442 third base redundancy, 347–48 threonine, 19 thymidylate synthetase, 109 thymine, 54–56, 60 tight mutation, 335 time-of-flight mass spectrometry, 722–23, 725 Ti-plasmids, 447–50 tmRNA, 217–18 TMV (tobacco mosaic viruses), 470–71, 473 Tn196 transposon, 420 TNV (tobacco necrosis virus), 480 tobacco mosaic viruses (TMV), 470–71, 473 tobacco necrosis virus (TNV), 480 topoisomerase IV, 106–7, 123 topoisomerases, 89–90 topoisomers, 90, 92 totipotent plants, 45 Tra+, 496 tra genes, 496 transcription, 133 in archaebacteria, 28 attenuation of, 297–99 basic components in, 134 in central dogma, 65 differing patterns of, 66 783 enhancers, 152–53 in eukaryotes, 145–46 of protein-encoding genes, 148–51 of ribosomal RNA (rRNA), 146–47 start of, 707–9 of transfer RNA (tRNA), 146–47 transcription bubble, 137 transcription-coupled repair, 387–88, 389 transcription factors, 264 independent domains of, 265 types of, 146 transcriptome, 694, 709 transcripts, 133 transduction, 485, 493 generalized, 493–94 specialized, 494–95 transfection, 489–90 transferases, 182 transfer RNA (tRNA), 67–69; see also RNA altered decoding by, 362–63 charging with amino acids, 204, 205 cloverleaf structure of, 200–201 initiator, 210 modified bases in, 201–2, 203 processing of, 305, 306 recognition of codons by, 200, 202–4 sites for, 211–13 suppressor, 363 transcription of, 146–47 transformation, 487–88 DNA as genetic material, 488–91 gene transfer by, 488 in nature, 491–93 transgenic organisms, 700 transition state, 182 transition state analogs, 185 transition state energy, 182 translation, 28, 65, 198 translational activators, 287–88 translational regulation, 282; see also gene regulation by alterations to ribosomes, 290–91 anti-sense RNA in, 288–90 degradation of mRNA, 282–83 regulatory proteins in, 284–87 riboswitches in, 299–300 translational repression, 284–86 translatome, 718–19 translocases, 222, 226 translocations, 213, 335, 343–45 transport proteins, 74, 174 transposable elements, see transposons transposases, 398, 403 transpositions, 397 abortive, 410 conservative, 401–7 in rearrangement of host DNA, 408–9 replicative, 402–7 transposons, 397 bacteriophage Mu, 417–20 complex, 402 784 Index components of, 399 composite, 406–9 conjugative, 420–21 essential parts of, 398–400 in higher life forms, 410–12 homing introns, 423–24 in insertion mutations, 341–42 insertion sequences, 400–401 integrons, 420–22 junk DNA, 422–23 in mobile DNA, 397–98 movements, 401–6 conservative transposition, 401, 402–3 replicative transposition, 402–6 properties of, 399 replication of, 50 retro-elements, 412–14 retrons, 416–17 selfish DNA, 422–23 trans-splicing, 318 TRAP (tryptophan attenuation protein), 299 trimethoprim, 109, 442 triploidy, trisomy, tRNA, see transfer RNA true revertants, 359–61 Trypanosoma, 128 trypanosomes, 520–25 trypsin, 180 tryptophan attenuation protein (TRAP), 299 tsetse fly, 523 tumor-DNA (T-DNA), 448–50 Tus protein, 122 twintrons, 315 twist, 89 two-component regulatory system, 253–54 two-hybrid analysis, 732–37; see also proteomics mass screening by mating, 738 principle of, 735 vectors for, 736 Ty1 element, 412–13 type-C virogene, 565 type II restriction enzymes, 603, 604, 716 type I restriction enzymes, 602–3 U U2 accessory factor (U2AF), 312 ubiquitin, 233 ultraviolet light, 582–83 ultraviolet radiation, 350 umber, 363 unequal crossing over, 83 universal genetic code, 227 upstream elements, 149, 151–52 upstream region, 135 deletion analysis of, 702–3 protein binding sites, 702–5 uracil, 54–56, 349 uracil-N-glycosylase, 384 urea, 196 uridine triphosphate (UTP), 587 urkaryotes, 554–55 U-RNA, 304 UTP (uridine triphosphate), 587 UvrAB excision repair system, 381–83 UV (ultraviolet) light, 582–83 V vaccination, 48, 468 Vaccinia, 468 van der Waals forces, 165 variable number of tandem repeats (VNTRs), 84 variant surface glycoprotein (VSG), 520–26 vectors, 608 bacteriophage lambda, 616–18 cosmid, 617–19 detecting insertions in, 612–14 exon trapping, 690 expression, 631–33 inserting genes into, 610–12 multicopy plasmids, 610 plasmids, 33 shuttle, 615–16 for two-hybrid analysis, 736 vegetative reproduction, 104, 485 Venter, Craig, 681 vertical gene transfer, 564 Vibrio cholerae, 428 viral diseases, 48 virions, 46, 454 virogene, 565 viroids, 480–81 characteristics of, 49, 479 coconut cadang-cadang, 481 replication of, 482 self-cleavage of, 542 structure of, 481 virtual masks, 712 virulence factors, 446 virulence plasmids, 442, 446–47 viruses, 454–55 bacterial, see bacteriophages characteristics of, 46–47 diversity of, 462—63 DNA-containing, 466–67 early genes of, 457 entry into cells, 48 families of, 463 genomes, 458, 462 genome sizes, 79 late genes of, 457 latency of, 460–62 life cycle of, 455–57 lysogeny of, 460–62 lytic growth of, 451–52 prions, 481–83 retroviruses, 472–77 RNA-containing, 467–69 rolling circle replication, 433 satellite, 480 structural components of, 46 structures of, 464 subcellular genetic elements, 49–50 synthesis and assembly of, 458 viroids, 480–81 virusoids, 479 vitamins, 172 VMA1 gene, 729 VNTRs (variable number of tandem repeats), 84 VSG (variant surface glycoprotein), 520–26 W Watson, James, 58, 76–77 Western blotting, 594–95, 722, 724 wild-type alleles, wild-type genes, Wilkins, Maurice, 77 wobble rules, 202 Wohler, Friedrich, 76 writhe, 89 X X chromosomes, 13–15, 277–80 X-gal, 185, 612, 696 X-inactivation, 277–80 Xis protein, 375–76 Xist gene, 278, 279 X-phos, 588, 696 X-ray crystallography, 171 Y YAC (yeast artificial chromosome), 620, 681, 683 Y chromosomes, 14–15 and African Eve hypothesis, 560–61 of Gengis Khan, 563 yeasts, 40–41 2-micron plasmids of, 450–51 haploid and diploid phases of, 527 mating types, 525–30 retrotransposons of, 412–13 two-hybrid system, 732–37 Yersinia pestis, 443, 447 Yorktown Technologies, 43 Z Z-DNA, 91, 93–94 zebrafish, 42–44 ZFY gene, 560 zinc finger, 193–94 Zipcode sequences, 688 zoo blotting, 595 zwitterion, 155 zygotes, 12, 511 ... heredity—in other words, the term molecular biology is often substituted for the perhaps more appropriate term, molecular genetics A more broad-minded definition of molecular biology includes all aspects... resulting revelation of the molecular basis of inheritance has resulted in the increasing use of the term molecular. ” Often the term molecular biology refers to the biology of those molecules... Cataloging-in-Publication Data Application submitted British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library ISBN: 0-1 2-1 7555 1-7 For