Methods 643

Techniques of Molecular Biology 647 INTRODUCTION 647 NUCLEIC ACIDS 648

Electrophoresis through a Gel Separates DNA and RNA Molecules According to Size 648 Restriction Endonucleases Cleave DNA Molecules at Particular Sites 649 DNA Hybridization Can Be Used to Identify Specific DNA Molecules 651 Hybridization Probes Can Identify

FJectrophorettcally-Separated DNA s and RNAs 652 Isolation of Specific Segments of DNA 653 DNA Cloning 654 Cloning DNA in Plasmid Vectors 654 Vector DNA Can Be Introduced into Host Organisms by Transformation 655 Libraries of DNA Molecules Can Be Created by Cloning 656 Hybridization Can 13e Used to Identify7 a Specific

Clone m a DNA Library 657 Chemically Synthesized Oligonucleotides 657 The Polymerase Chain Reaction (PCR) Amplifies DN As by Repeated Rounds of DNA Replication in vitro 658 Nested Sets of DNA Fragments

Reveal Nucleotide Sequences 660 Box 20-1 Foremics and the Polymerase Chain Reaction 661

Shotgun Sequencing a Bacterial Genome 663 The Shotgun Strategy Permits a Partial Assembly of Large Genome Sequences 664 Box 20-2 Sequemitors Are Used far High Throughput Sequencing 665

The Paired-End Strategy Permits the Assembly of Large Genome Scaffolds 666 Genome-Wide Analyses 667 Comparative Genome Analysis 669 PROTEINS 672 Specific Proteins Can Be Purified from Cell Extracts 672

Purification of a Protein Requires a Specific Assay 673 Preparation of Cell Extracts Containing Active Proteins 673 Proteins Can Be Separated from One Another Using

Column Chromatography 673 Affinity Chromatography Can Facilitate More R;-ipid

Protein Purification 674 Separation of Proteins on Polyacrylamide Gels 675 Antibodies Visualize Eleeirophoretically-Separated

Proteins 676 Protein Molecules Can Be Directly Sequenced 676 Proteomics 677 Bibliography 679

xxvi Detailed Contents

CHAPTER 21 Model Organisms 681 BACTERIOPHAGE 682

Assays of Phage Growth 684 The Single-Step Growth Curve 685 Phage Crossses and Complementation Tests 685 Transduction and Recombinant DNA 686 BACTERIA 687 Assays of Bacterial Growth 687 Bacteria Exchange DNA by Sexual Conjugation, Phage-Mediated Transduction, and DNA-Mediated Transformation 688 Bacterial PI asm ids Can Be Used as Cloning Vectors 689 Transposons Can Be Used to Generate lnsertional Mutations and Gene and Operon Fusions 689 Studies on the Molecular Biology of Bacteria Have Been Enhanced by Recombinant DNA Technology, Whole-Genome Sequencing, and Transcriptional Profiling 690 Biochemical Analysis Is Especially Powerful in Simple Cells with Well-Developed Toots of Traditional and Molecular Genetics 691 Bacteria Are Accessible to Cytological Analysis 691 Phage and Bacteria Told Us Most of the Fundamental Things about the Gene 692 BAKER'S YEAST, Sticcharomyces cerevisiae 693 The Existence of Haploid and Diploid Cells Facilitate

Genetic Analysis of S. cercvisiae 693 Generating Precise Mutations in Yeast Is Easy 694 S. cereinsiae Has a Small, Well-Characterized Genome 694 S. cerevisiae Cells Change Shape as They Grow 695

THE NEMATODE WORM, Caenorhabdiiis etegmts 696

C. elegpm Has a Very Rapid Life Cycle 696 C. eiegans Is Composed of Relatively Few, Well Studied Cell Lineages 697 The Cell Death Pathway Was Discovered in C. elegans 698 RNAi Was Discovered in C. elegans 698 THE FRU IT FLY, Drosopfula melanogaster 699 Drosopfula Has a Rapid Life Cycle 699 The First Genome Maps Were Produced in Drosophiia 700 Genetic Mosaics Permit the Analysis of Lethal Genes in Adult Flies 702 The Yeasr FLP Recombinase Permits the Efficient

Production of Genetic Mosaics 703 It Is Easy to Create Transgenic Fruit Flies that Carry Foreign DNA 703 THE HOUSE MOUSE, Mus mwscutus 705 Mouse Embryonic Development Depends on Stem Cells 706 It Is Easy to Introduce Foreign DNA

into the Mouse Embryo 707 Homologous Recombination Permits the Selective Ablation of Individual Genes 707 Mice Exhibit Epigenetic Inheritance 709 Bibliography 711

Index 713

Class Testers and Reviewers

We wish to thank all of the instructors tor their thoughtful suggestions and comments, including;

Chapter Reviewers

Ann Aguanrio, Man'movnt Manhattan College Charles F. Austerberry, Creighton University David G. Rear, University of New Mexico

Health Sciences Center Margaret E. Beard, Holy Cross Call S. Beg ley, Northeastern University Sail ford Bernstein, San Dingo State University Michael Blaher, Florida State University Nicole Efournias, California State University,

San Bernardino John Boyle, Mississippi State University Suzanne Rradshaw, University of Cincinnati John G. Burr, f diversity of Texas at Dallas Michae! A. Campbell, Pennsylvania State

I University, Erie, The Behrend College Shirley Goomber, king's College, University of London Anne Cordon, University of Toronto Sumana Datla, Texas A&M University Jeff Dejong, University of Texas at Dallas furgon Denecke, University of Leeds Susan M. DiBartolomcip, Milleraville University Santosh R. D'Mello, University of Texas at Dallas Robert Duronio, University of North Carolina,

Chapel Hill Steven W.Edwards, University of Liverpool Allen Galhman, Southeast Missouri State University Anthony D. M. Glass, University of British Columbia Elliott S, Goldstein, Arizona State University Ann Grens. Indiana University, South Bend Gregory B. Hecht, Hoivan University Robert R. Helling, University of Michigan David C. Higgs, LIniversity of Wisconsin, Parkside Mark Kainz, Colgate University Gregory M. Kelly, University of Western Ontario Ann Kleinschmidt, Allegheny College Dan Krane, Wright State University Mark Levinthal, Purdue University Gary J. Lindqucster, Rhodes College

Curtis Loer, University of San Diego

Virginia McDonough, Hope College

Michael J, Mcpherson. University of Leeds

Victoria Meiler, Tufts University

William L. Miller, North Carolina State University

Dragana Miskovic, University of Waterloo

David Mullin, Tulane University

Jeffrey D. Newman, Lycoming College

James B, Olesen, Ball State University

Anthony J. Olsuka, Illinois State University

Karen Palter, Temple University

James G. Patton, Van derbilt University

Jan R. Phillips, Queen Mary, University of London

Steve Picksley, University of Bradford

Todd P. Primm, University of Texas at El Paso

Eva Sapi, University of New Haven

Jon R. Scales, Midwestern State University

Michael Schnitze, University of York

Venkal Sharma, University of Hfeit Elorida lirica L. Shelley, University of Toronto at Mississaugo

Elizabeth A. Shephaid, University College. London

Maigaret E. Stevens, Kipon College

Akif Uzman, University of Houston, Downtown

Quinn Vega, Montclair State University

Jeffrey M. Voight, Alhuny College of Pharmacy

Robert Wiggers, Stephen F. Austin State University

Bruce C. Wight man, Muhlenberg College

Class Testers

Charles F. Austerberry, Creighton University Chrisline E. Bezotte, Elmira College Astrid Helfant, Hamilton College Gerald Joyce, The Scripps Research Institute Jocclyn Krebs, University of Alaska, Anchorage Cran Lucas, Louisiana State University in Shieveporl Anthony J. Ofsuka, Illinois State University Charles Poison, Florida institute of Technology Ming-Che Shih, University of Iowa

About the CD and Website

The student CD-ROM for Molecular Biology of the Gene provides resources to help students visualize difficult concepts, explore complex processes, find review their understanding of the most challenging material presented in this coursc. This easy to use electronic resource provides students with rapid access to twenty interactive tutorials, thirteen structural animations, and Critical thinking exercises that can be assigned by instructors, The tutorials contain animations that are broken out step by step, so that students can focus on mastering one element at a time. Every tutorial concludes with an "Apply Your Knowledge" activity, where students are presented with a problem and then guided through to the solution with interactive animations and multiple choice questions. The structural animations run in ClilME, an application that automatically converts the information needed to define the three-dimensional structures of many molecules into accurate molecular models and presents it in a window in your Netscape Navigator browser. Finally, the critical thinking activities ask students to actively engage with the materia].

The student website for Molecular Hioiogy of the Gene also provides the twenty interactive tutorials, fifteen structural animations, and critical thinking exercises found on the CD-ROM, but also contains additional research tools and web resources that are outstanding tools for students wishing to explore a chapter's concepts or extend their knowledge beyond the scope of the text. In combination with the student CD, the student website provides a valuable set of resources to help students develop the skills they need to succeed in class.

DNA TOPOLOGY TWJST & WRiTHF

Objectives,

Objectives,

► Understand wtiy the (inking number for a molecule of co^alentiy ci osed circular DMA (cccONA) is constant

part

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