Molecular Biology

802 Pages·2005·36.72 MB·English

MMOOLLEECCUULLAARR
BBIIOOLLOOGGYY
David Clark
Southern Illinois University
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MOLECULAR
BIOLOGY

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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 Revolu- cell biology and so can pick and choose from the topics
tion,reflects the massive surge in our understanding of the covered as needed. However, others will not be so well
molecular foundations of genetics in the last fifty years.In prepared,due in part to the continuing influx into molec-
the next half century our understanding of how living ular biology of students from related disciplines.For them
organisms function at the molecular level, together with I’ve tried to create a book whose early chapters cover the
our ability to intervene,will expand in ways we are only basics,before launching out into the depths.
just beginning to perceive. Because of the continuing interest in applying molec-
Today we now know that genes are much more than ular biology to an ever widening array of topics, I have
the abstract entities proposed over a century ago by tried to avoid overdoing detail (depth) in favour of
Mendel.Genes are segments of DNA molecules,carrying breadth.This in no way minimizes the importance of the
encoded information. Indeed, genes have now become subject matter for cell biologists but instead emphasizes
chemical reagents,to be manipulated in the test tube.In that molecular biology is applicable to more than just
the days of classical genetics genes represented inherited human medicine and health.The genetic revolution has
characteristics but were themselves inviolate, rather like also greatly impacted other important areas such as agri-
atoms before the twentieth century.Today both genes and culture,veterinary medicine,animal behaviour,evolution,
atoms have sub-components to be tinkered with. and microbiology. Students of these, and related disci-
A full understanding of how living organisms function plines,all need to understand molecular biology at some
includes an appreciation of how cells operate at the level.
molecular level.This is of vital importance to all of us as Finally there are no references or extra reading at the
it becomes ever more clear that molecular factors under- ends of the chapters,for two reasons.My own cross-ques-
lie many health problems and diseases.While cancer is the tioning has revealed that neither myself nor most of my
“classic”case of a disease that only became understand- colleagues and students have ever actually used such text-
able when its genetic basis was revealed,it is not the only book references just as we rarely watch the extra material
one by any means.Today the molecular aspects of medi- on DVDs providing actors’ insights,extra scenes,and out-
cine are expanding rapidly and it will soon be possible to takes. The student has enough to deal with in the core
personally tailor clinical treatment by taking into account material.
the genetic make-up of individual patients. Secondly, anyone who wants up-to-date reference
Rather than attempting to summarize my view of material is far better advised to run a web search.PubMed,
modern molecular biology (the book itself,I hope,accom- Google Scholar and Scirus.com are good choices.
plishes that) in a short preface,I’d like to briefly address Feedback (hopefully positive!) is welcome.
what this book is not.It is not intended as a reference work
for faculty or researchers but rather as a survey-oriented David Clark,Carbondale,Illinois,January 2005
textbook for upper division students in a variety of bio-
logical sub-disciplines.In particular it is intended for final
year undergraduates and beginning graduate students. Acknowledgements
This book does not attempt to be exhaustive in its
coverage, even as a textbook.There is a second book in I would like to thank the following individuals for their
this series (Biotechnology:Applying the Genetic Revolu- help in providing information, suggestions for improve-
tion, 2006) co-authored with Nanette Pazdernik, which ment and encouragement: Laurie Achenbach, Rubina
essentially picks up where this book ends.Both books,I Ahsan, Phil Cunningham, Michelle McGehee, Donna
hope,effectively survey the foundations and applications Mueller,Dan Nickrent,Joan Slonczewski.Especial thanks
of modern molecular genetics. go to Nanette Pazdernik for help in editing many of the
Many,perhaps most,of the students using this book chapters and to Karen Fiorino for creating most of the
will be well versed in the basics of modern genetics and artwork.
vii
Introduction
Molecular Genetics Is and analyzing the vast mass of genetic information con-
stantly emerging from experimentation requires the use
Driving the Biotechnology
of sophisticated software and powerful computers. The
Revolution
emerging information revolution rivals the industrial rev-
olution in its importance,and the consequences of today’s
Although the breeding of plants and animals goes back findings are already changing human lives and will con-
thousands of years,only in the last couple of centuries has tinue to alter the lives of future generations.Data is accu-
genetics emerged as a field of scientific study. Classical mulating about the molecules of inheritance and how they
genetics emerged in the 1800s when the inheritance pat- are controlled and expressed at an ever faster and faster
terns of such things as hair or eye color were examined pace.This is largely due to improved techniques,such as
and when Gregor Mendel performed his famous experi- PCR (polymerase chain reaction; see Ch. 23) and DNA
ments on pea plants.Techniques revealing how the inher- (deoxyribonucleic acid) arrays (see Ch.25).In particular,
ited characteristics that we observe daily are linked to methods have recently been developed for the rapid,
their underlying biochemical causes have only been devel- simultaneous and automated analysis of multiple samples
oped since World War II.The resulting revelation of the and/or multiple genes.
molecular basis of inheritance has resulted in the increas- One major impact of molecular biology is in the realm
ing use of the term “molecular.”Often the term “molecu- of human health. The almost complete sequence of the
lar biology” refers to the biology of those molecules DNA molecules comprising the human genome was
related to genes, gene products and heredity—in other revealed in the year 2003.So,in theory,science has avail-
words,the term molecular biology is often substituted for able all of the genetic information needed to make a
the perhaps more appropriate term,molecular genetics.A human being.However,the function of most of a human’s
more broad-minded definition of molecular biology approximately 35,000 genes remains a mystery.Still more
includes all aspects of the study of life from a molecular complex is the way in which the expression of these genes
perspective. Although the molecular details of muscle is controlled and coordinated.Inherited diseases are due
operation or plant pigment synthesis could be included to defective versions of certain genes or to chromosomal
under this definition,in practice,textbooks are limited in abnormalities.To understand why defective genes cause
length.In consequence,this book is largely devoted to the problems, it is important to investigate the normal roles
molecular aspects of the storage and transmission of bio- of these genes.As all disease has a genetic component,the
logical (i.e.,genetic) information. present trend is to redefine physical and mental health
Although there is great diversity in the structures and from a genetic perspective.Even the course of an infec-
lifestyles of living organisms,viewing life at the molecular tious disease depends to a significant extent on built-in
level emphasizes the inherent unity of life processes. host responses,which are determined by host genes.For
Perhaps it is this emergent unity, rather than the use of example,humans with certain genetic constitutions are at
sophisticated molecular techniques,that justifies molecu- much greater risk than others of getting SARS, even
lar biology as a discipline in its own right. Instead of an though this is an emerging disease that only entered the
ever-expanding hodge-podge of methods for analyzing human population in the last few years.The potential is
different organisms in more and more detail, what has present to improve health and to increase human and
been emerged from molecular analysis is an underlying animal life spans by preventing disease and slowing the
theme of information transmission that applies to all life aging process. Clinical medicine is changing rapidly to
forms despite their outward differences. incorporate these new findings.
Society is in the midst of two scientific revolutions. The other main arena where biotechnology will have
One is in the realm of technology of information,or com- a massive impact is agriculture. New varieties of geneti-
puters, and the other in molecular biology. Both are cally engineered plants and animals have already been
related to the handling of large amounts of encoded infor- made and some are in agricultural use.Animals and plants
mation. In one case the information is man made, or at used as human food sources are being engineered to adapt
any rate man-encoded,and the mechanisms are artificial; them to conditions which were previously unfavorable.
the other case deals with the genetic information that Farm animals that are resistant to disease and crop plants
underlies life. Biology has reached the point where the that are resistant to pests are being developed in order to
genes that control the makeup and functioning of all living increase yields and reduce costs. The impact of these
creatures are being analyzed at the molecular level and genetically modified organisms on other species and on
can be altered by genetic engineering. In fact, managing the environment is presently a controversial issue.
viii
Table of Contents
CHAPTER 1 Basic Genetics 1
CHAPTER 2 Cells and Organisms 21
CHAPTER 3 DNA,RNA and Protein 51
CHAPTER 4 Genes,Genomes and DNA 75
CHAPTER 5 Cell Division and DNA Replication 103
CHAPTER 6 Transcription of Genes 132
CHAPTER 7 Protein Structure and Function 154
CHAPTER 8 Protein Synthesis 197
CHAPTER 9 Regulation of Transcription in Prokaryotes 234
CHAPTER 10 Regulation of Transcription in Eukaryotes 262
CHAPTER 11 Regulation at the RNA Level 281
CHAPTER 12 Processing of RNA 302
CHAPTER 13 Mutations 333
CHAPTER 14 Recombination and Repair 368
CHAPTER 15 Mobile DNA 396
CHAPTER 16 Plasmids 425
CHAPTER 17 Viruses 453
CHAPTER 18 Bacterial Genetics 484
CHAPTER 19 Diversity of Lower Eukaryotes 508
CHAPTER 20 Molecular Evolution 533
CHAPTER 21 Nucleic Acids:Isolation,Purification,Detection,and
Hybridization 567
CHAPTER 22 Recombinant DNA Technology 599
CHAPTER 23 The Polymerase Chain Reaction 634
CHAPTER 24 Genomics and DNA Sequencing 662
CHAPTER 25 Analysis of Gene Expression 693
CHAPTER 26 Proteomics:The Global Analysis of Proteins 717
Glossary 745
Index 771
ix
Detailed Contents
CHAPTER 1 Basic Genetics 1 Some Widely Studied Organisms Serve as
Models 40
Gregor Mendel Was the Father of Yeast Is a Widely Studied Single-Celled
Classical Genetics 2 Eukaryote 40
Genes Determine Each Step in Biochemical A Roundworm and a Fly are Model
Pathways 3 Multicellular Animals 41
Mutants Result from Alterations in Genes 4 Zebrafish are used to Study Vertebrate
Phenotypes and Genotypes 5 Development 42
Chromosomes Are Long,Thin Molecules Mouse and Man 44
That Carry Genes 6 ArabidopsisServes as a Model for Plants 44
Different Organisms may Have Different Haploidy,Diploidy and the Eukaryote Cell
Numbers of Chromosomes 7 Cycle 45
Dominant and Recessive Alleles 8 Viruses Are Not Living Cells 46
Partial Dominance,Co-Dominance, Bacterial Viruses Infect Bacteria 47
Penetrance and Modifier Genes 9
Human Viral Diseases Are Common 48
Genes from Both Parents Are Mixed by
A Variety of Subcellular Genetic Entities Exist 49
Sexual Reproduction 11
Sex Determination and Sex-Linked
CHAPTER 3 DNA, RNA and
Characteristics 13
Protein 51
Neighboring Genes Are Linked during
Inheritance 15
Nucleic Acid Molecules Carry Genetic
Recombination during Meiosis Ensures
Information 52
Genetic Diversity 16
Chemical Structure of Nucleic Acids 52
Escherichia coliIs a Model for Bacterial
DNA and RNA Each Have Four Bases 54
Genetics 17
Nucleosides Are Bases Plus Sugars;
CHAPTER 2 Cells and Organisms 21 Nucleotides Are Nucleosides Plus Phosphate 55
Double Stranded DNA Forms a Double Helix 56
What Is Life? 22
Base Pairs are Held Together by Hydrogen
Living Creatures Are Made of Cells 23
Bonds 57
Essential Properties of a Living Cell 23
Complementary Strands Reveal the Secret of
Prokaryotic Cells Lack a Nucleus 27 Heredity 59
Eubacteria and Archaebacteria Are Constituents of Chromosomes 60
Genetically Distinct 28
The Central Dogma Outlines the Flow of
Bacteria Were Used for Fundamental Studies Genetic Information 63
of Cell Function 29
Ribosomes Read the Genetic Code 65
Escherichia coli (E.coli)Is a Model Bacterium 31
The Genetic Code Dictates the Amino Acid
Where Are Bacteria Found in Nature? 32 Sequence of Proteins 67
Some Bacteria Cause Infectious Disease,but Various Classes of RNA Have Different
Most Are Beneficial 34 Functions 69
Eukaryotic Cells Are Sub-Divided into Proteins,Made of Amino Acids,Carry Out
Compartments 34 Many Cell Functions 70
The Diversity of Eukaryotes 36 The Structure of Proteins Has Four Levels of
Eukaryotes Possess Two Basic Cell Lineages 36 Organization 71
Organisms Are Classified 38 Proteins Vary in Their Biological Roles 73
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