Insect Molecular Genetics. An Introduction to Principles and Applications

545 Pages·1994·7.628 MB·English

by  Marjorie A. Hoy (Auth.)

Insect Molecular
Genetics
An Introduction to
Principles and
Applications
Marjorie A* Hoy
Entomology and Nematology Department
Institute of Food and Agricultural Sciences
University of Florida
Gainesville, Florida
Academic Press
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Library of Congress Cataloging-in-Publication Data
Hoy, Marjorie A.
Insect molecular genetics / by Marjorie A. Hoy
p. cm.
Includes bibliographical references and index.
ISBN 0-12-357490-0
1. Insects-Molecular genetics. I. Title.
QL493.H69 1994
595.7'87328-dc20 94-7475
CIP
PRINTED IN THE UNITED STATES OF AMERICA
94 95 96 97 98 99 BB 9 8 7 6 5 4 3 2 1
Preface
The development of recombinant DNA techniques during the past 20 years has
resulted in exciting advances in the detailed study of specific genes at the
molecular level as well as breakthroughs in molecular, cellular, and developmen­
tal biology. Of the molecular genetics studies conducted on insects, most have
been directed to Drosophifo mefonogaster. Relatively few data have been gener­
ated by molecular biological methods from analyses of other insects. Yet, the
application of molecular genetics to insects other than Drosophifo has the poten­
tial to revolutionize insect population and organismal biology.
Why have molecular genetic techniques been used so little by entomolo­
gists? There may be a number of reasons. Recombinant DNA techniques are
most readily carried out by people trained in biochemistry and relatively few
entomologists are so trained. The techniques have been, until recently, relatively
complex and difficult, so that strong technical skills were required. Also, most
entomologists have been slow to ask whether these techniques were appropriate
for studies of population or organismal biology because much of the published
literature has focused on fundamental issues of Drosophifo gene structure, regula­
tion and function, developmental regulation, and evolution.
Goals
My goal is to introduce entomologists to the concepts of molecular genetics
without assuming that they have received previous training in molecular biology.
This book is not intended to substitute for formal training in biochemistry or
molecular genetics. If novice readers wish to develop molecular genetics skills,
they must obtain additional training in genetics and biochemistry. However, the
book will provide an introduction to terminology, as well as an overview of
principles, techniques, and possible applications of molecular genetics to prob­
lems of interest to entomologists.
XX Preface
In preference to using examples from the Drosophûa literature, I have used
examples in which other arthropods have been studied. However, without
doubt, Drosophila is the premier model for insect molecular genetics study. One
fond hope is that this book will be a bridge for entomologists seeking to apply the
exciting methods developed for Drosophih and that it will introduce Drosophila
workers to some of the problems and issues of interest to entomologists seeking
to solve applied problems. Perhaps this book will help to break down the barriers
between entomologists and Drosophila workers isolated from each other by per-
spective and technical jargon. If this book helps to achieve these goals, it will
have served its purpose.
Organization
The book was designed for a one-semester course in insect molecular genetics for
upper division undergraduates or beginning graduate students. The initial por-
tion of the book reviews basic information about DNA, RNA, and other impor­
tant molecules (Chapters 1-4). Readers with a recent course in genetics could
skip this section. Chapter 5 describes the genetic systems found in insects and
gives an overview of development sufficient to understand subsequent topics
such as P-element-mediated transformation and sex determination. Chapters 6-
10 provide introductions to useful techniques, including cloning, library con­
struction, sequencing, the polymerase chain reaction, and P-element-mediated
transformation of Drosophih. Most molecular biologists reading this book could
skip this section as well. Chapters 6-10 are not intended as a laboratory manual
but, in some cases, an outline of laboratory protocols is provided in order to
furnish the novice with a sense of the complexity or simplicity of the procedures
and some of the issues to consider in problem solving. Throughout the book,
references are provided for the reader interested in pursuing specific topics and
techniques, although they are not exhaustive. Despite the value of providing an
historical overview, I have not always provided references to the first publication
on a subject. Rather, review articles or recent publications that include refer­
ences to earlier work are cited.
Finally, in the third section of the book (Chapters 11-16), I have attempted
to demonstrate how molecular genetic techniques can solve a diverse array of
basic and applied problems. Part III is intended to introduce readers to the
exciting molecular research that is revolutionizing insect biology, ecology, sys-
tematics, behavior, physiology, development, sex determination, and pest man­
agement. Each chapter in this section could be read by itself, assuming that the
reader understands the appropriate concepts or information presented in Parts I
and II.
Each chapter begins with an overview or brief summary of the material
being covered. The overview should be read both before and after reading each
Preface xxi
chapter to review the concepts covered. The overview is followed by a brief
introduction covering the history or rationale for the topic. References at the
end of the chapter are provided for further reading. Where possible, books or
reviews are cited to provide an entry into the literature. Recent references are
provided, but no attempt has been made to review all the literature on a specific
topic. Simple protocols may be given to provide the flavor of specific techniques,
although these are not intended to be complete. References to handbooks or
technique books are also provided at the end of appropriate chapters. When a
term that may be unfamiliar is first introduced, it is in bold type and a brief
definition or description is given in the Glossary at the end of the book. Finally,
in Appendix I, a time line of some significant advances in genetics, molecular
biology, and insect molecular genetics provides a perspective of the pace with
which dramatic advances have been, and continue to be, made.
Progress is rapid in molecular genetics, and this book can only provide an
introduction to the principles of insect molecular genetics and some of its
applications. It is impossible to provide a complete review of the insect molecu­
lar genetics literature in a book of this size. The literature cited includes refer­
ences through 1993 and focuses on genetics. It is not intended to be an in­
troduction to all aspects of 'molecular entomology,' which has been defined as
"... a blend of insect science, molecular biology, and biochemistry." The divid­
ing line between molecular entomology and insect molecular genetics is some­
times difficult to resolve.
Shortly before this book went to the publisher, two related books were
published: "Molecular Approaches to Fundamental and Applied Entomology"
edited by J. Oakeshott and M. J. Whitten, and "Insect Molecular Science" edited
by J. M. Cramp ton and P. Eggleston. Both multiauthored books cover some of
the topics included here, but assume the reader is familiar with molecular genet­
ic techniques and terminology; they would be daunting for the novice.
Acknowledgments
This book would not have been written without the encouragement of many
people. As most authors remark, it was more difficult to write than expected, in
part due to a move from the University of California at Berkeley to the Univer­
sity of Florida in Gainesville about half-way through the endeavor. Certainly,
this book would not have been written without the support of the Rockefeller
Foundation which provided me with five weeks of precious time at the Bellagio
Study and Conference Center in Italy. I gained a crucial grasp on the scope of
the project and gathered my courage there while on sabbatical from the Univer­
sity of California at Berkeley. A fellowship spent at Cold Spring Harbor Labora­
tories in the summer of 1985 in the 'Molecular Cloning of Eukaryotic Genes'
course provided my initial training in molecular genetic techniques. I thank the
xxu Preface
instructors for their patience. Likewise, G. M. Rubin kindly allowed me to
participate in his cloning course at the University of California at Berkeley.
Many people have contributed information, advice, and valuable time re­
viewing this book. I especially thank Mary Bownes, Michael Caprio, Gary Car-
valho, Howell Daly, Owain Edwards, Marilyn Houck, James Hoy, A.
Jeyaprakash, Srini Kambhampati, Carolyn Kane, James Presnail, Veronica
Rodriguez, Mark Tanouye, and Tom Walker, who reviewed either drafts of indi­
vidual chapters or the entire book. Richard Beeman, Owain Edwards, Glenn
Hall, A. Jeyaprakash, Ed Lewis, Jim Presnail, and A. Zacharopoulou provided
photographs or illustrations. Lois Caprio and Denise Johanowicz assisted with
many of the logistical issues. Finally, I especially thank my husband Jim, who
repeatedly motivated me to clarify and elucidate the principles and applications
presented here and tolerated my preoccupation with this project over many
months, and John Capinera, who patiently waited for me to emerge from my
compulsion. Despite the best efforts of the reviewers, errors probably persist and
are my responsibility. This is University of Florida, Institute of Food and Agri­
cultural Sciences, series number R-03561.
Marjorie A. Hoy
Chapter 1
DNA and Gene Structure
Overview
Arthropod genes are made of DNA and are located in chromosomes consist­
ing of proteins, RNA, and DNA. DNA is a polymer of nucleotides. Each nucle-
otide consists of a pentose sugar, one of four different nitrogenous bases, and a
phosphoric acid component. DNA consists of two complementary nucleotide
strands in a helix form. Pairing of the nitrogenous bases adenine (A) with
thymine (T), and cytosine (C) with guanine (G) on the two complementary
strands occurs by hydrogen bonding. A pairs with T by two hydrogen bonds, and
C pairs with G by three hydrogen bonds. DNA has chemically distinct 5 ' and 3 '
ends. The two strands are antiparallel, with one running in the 5 ' to 3 ' direction
and the other from the 3 ' to 5 ' direction. Genetic information is determined by
the sequence of nitrogenous bases (A, T, G, C) in one of the strands, with a
three base (triplet) codon designating an amino acid. The genetic code is degen­
erate, with more than one codon specifying most amino acids. The genetic
information is expressed when DNA is transcribed into messenger RNA, which
is translated into polypeptides. Most arthropod genes have intervening se­
quences of noncoding nucleotides (introns) that must be removed from the
primary RNA molecule before translation can occur.
Introduction to the Central Dogma
The Central Dogma, as proposed by Francis Crick in 1958, stated that
biological information is carried in DNA; this information is transferred first to
RNA, and then to proteins. Initially, the Central Dogma also stated that the
flow of information is unidirectional, with proteins unable to direct synthesis of
RNA, and RNA unable to direct the synthesis of DNA (Figure 1.1). However,
the Central Dogma was amended in 1970 when certain viruses were found to
3
4 Chapter 1 DNA and Gene Structure
O
Replication
DNA
Transcription
RNA
Translation
PROTEIN
Figure 1.1 The Central Dogma. Biological information is transferred from DNA to
RNA to proteins. Recent discoveries of viruses that transcribe information from RNA to
DNA have required modification in the Dogma.
transfer information from RNA to DNA. Despite this modification, the Central
Dogma remains a major tenet of modern biology.
In insects, the genes (DNA) are found in complex structures called chromo­
somes that consist of proteins, RNA, and DNA. This chapter reviews the struc­
ture of DNA and RNA and the basis of the genetic code.
The Molecular Structure of DNA
Deoxyribonucleic acid (DNA) is a long polymeric molecule consisting of
numerous individual monomers that are linked in a series and organized in a
helix. Each monomer is called a nucleotide. Each nucleotide is itself a complex
molecule made up of three components: (1) a sugar, (2) a nitrogenous base, and
(3) a phosphoric acid.
5*
HÇ>CH ÇH
2
H ]3Τ-| · Η
2
HO H
2'-deoxyribose Ribose
Figure 1.2 Structure of sugars found in nucleic acids; 2'-deoxyribose is found in DNA
and ribose is found in RNA.
The Molecular Structure of DNA 5
PURINES
NH2
Κ
€ΊΓ
8C—H
Adenine Guanine
PYRIMIDINES
Î NH2
ï—CH3
■ 2 61
—H
H
Thymine Cytosine
Uracil
Figure 1.3 Bases in DNA are purines (adenine and guanine) or pyrimidines (thymine
and cytosine). Uracil is substituted for thymine in RNA.
In DNA, the sugar component is a pentose (with five carbon atoms) in a
ring form that is called 2'-deoxyribose (Figure 1.2). The nitrogenous bases are
single- or double-ring structures that are attached to the Γ-carbon of the sugar.
The bases are purines (adenine and guanine) or pyrimidines (thymine and
cytosine) (Figure 1.3). When a sugar is joined to a base it is called a nucleoside.
A nucleoside is converted to a nucleotide by the attachment of a phosphoric
acid group to the 5'-carbon of the sugar ring (Figure 1.4). The four different
6 Chapter 1 DNA and Gene Structure
5'-P terminus
O- O- Or
I II 5-
"O -p-o-p-o- P-0-ÇH2
0 00 > one nucleotide
<> I
A phosphodiester bond < o=P—O— CH2
I
o
OH
3'-OH terminus
Figure 1.4 A nucleoside consists of a sugar joined to a base. It becomes a nucleotide
when a phosphoric acid group is attached to the 5'-carbon of the sugar. Nucleotides link
together by phosphodiester bonds to form polynucleotides.
nucleotides that polymerize to form DNA are 2'-deoxyadenosine 5'-triphos-
phate (dATP or A), 2'-deoxyguanosine 5'-triphosphate (dGPT or G), 2'-deox-
ycytidine 5'-triphosphate (dCTP or C), and 2'-deoxythymidine 5'-triphosphate
(dTTP or T) (Figure 1.5). These names are usually abbreviated as dATP, dGTP,
dCTP, and dTTP, or even as A, G, C, and T
Individual nucleotides are linked together to form a polynucleotide by phos­
phodiester bonds, which are obtained by esterification of the two hydroxyl groups
of the same phosphoric acid molecule (Figure 1.4). Polynucleotides have chem­
ically distinct ends. In Figure 1.4, the top of the polynucleotide ends with a
nucleotide in which the triphosphate group attached to the 5' -carbon has not
participated in a phosphodiester bond. This is called the 5' or 5'-P terminus. At
the other end of the molecule, the unreacted group is not the phosphate, but the
3' -hydroxyl. This is called the 3' or 3' -OH terminus. This distinction between
the two ends means that polynucleotides have an orientation that is very impor­
tant in many molecular genetics applications.