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Dominic W. S. Wong
The ABCs
of Gene
Cloning
Third Edition
The ABCs of Gene Cloning
Dominic W. S. Wong
The ABCs of Gene Cloning

Third Edition
Dominic W. S. Wong
Western Regional Research Center
Albany, CA, USA
ISBN 978-3-319-77762-7 ISBN 978-3-319-77982-9 (eBook)

https://doi.org/10.1007/978-3-319-77982-9
Library of Congress Control Number: 2018937521
© Springer International Publishing AG, part of Springer Nature 1997, 2006, 2018
This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of
the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation,
broadcasting, reproduction on microfilms or in any other physical way, and transmission or information
storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology
now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication
does not imply, even in the absence of a specific statement, that such names are exempt from the relevant
protective laws and regulations and therefore free for general use.
The publisher, the authors and the editors are safe to assume that the advice and information in this book
are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the
editors give a warranty, express or implied, with respect to the material contained herein or for any errors
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Printed on acid-free paper
This Springer imprint is published by the registered company Springer International Publishing AG part
of Springer Nature.
The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland
To: Benji and Theo
Preface to the Third Edition
In preparing this third edition, the author has become convinced more
than ever that mastering the very basics of speaking and reading the “language”
of gene cloning is the key to see its beauty. The overall objective remains the
same as that stated for the two previous editions, with emphasis on the “nuts and
bolts” in learning the vocabulary and language of gene cloning. To this end, Part
I and II have included updates for the chapters on cloning techniques, cloning
vectors, and transformation. A new chapter is written on the concept and
approach in developing gene-vector constructs for expression cloning.
During the 12 years since the second edition was prepared, there has
been remarkable advancement in the application technology of gene cloning. In
revising this book, topics of emerging impact have been added, particularly
relating to the field of medical science and technology. Some of the new sec-
tions include: disease gene identification by exome sequencing, recombinant
Adeno- associated virus-mediated gene therapy, engineered nucleases and
CRISPR for gene/genome editing, and next generation sequencing. Other chap-
ters have been revised and updated as well.
It has been a delightful and inspiring experience to learn about the con-
tribution of numerous scientists to the ever-advancing field of gene cloning. I
should thank the authors whose publications and materials are referenced in this
book, and the publishers for giving permissions to use the copyrighted materi-
als. Thanks are due to many of my colleagues and students for the years of
research collaborations giving focus and meaning to the scope and presentation
of this book.
vii
Preface to the Second Edition
In the 9 years since the First Edition, my contention remains that an

effective approach to understand the subject of gene cloning is by learning the
“vocabulary” and the “language”. This book emphasizes the nuts and bolts on
just how to do that – reading and speaking the language of gene cloning. It
shows the readers how to distinguish between a gene and a DNA, to read and
write a gene sequence, to talk intelligently about cloning, to read science news
and to enjoy seminars with some degree of comprehension.
On the whole, the second edition is not any more advanced than the
first, with the intent of keeping the book concise and not burdening the readers
with unwarranted details. Nevertheless, changes were needed and new materials
were incorporated in the revision. Part I has a new chapter to provide a tutorial
on reading both prokaryotic and eukaryotic gene sequences. Part II consists of
several additions, updating on new techniques and cloning vectors. The topics
in Part III have been rearranged in separate sections – Part III now focuses on
applications of gene cloning in agriculture, and Part IV is devoted entirely to
applications in medicine. Chapters on gene therapy, gene targeting, and DNA
typing have been thoroughly revised. Additional coverage is included on animal
cloning and human genome sequencing. The heavy activity in rewriting and
expanding Part IV reflects the rapid progress in the technology and the increased
impact of gene cloning.
I enjoyed writing and revising this book with deep satisfaction. It has
been an inspiring experience to witness the remarkable development in the field
of gene cloning and the tireless dedication of thousands of scientists in making
genes tick.
ix
Preface to the First Edition
Gene cloning has become a fast growing field with a wide-ranging

impact on every facet of our lives. The subject of gene cloning could be intimi-
dating to the novice with little formal training in biology. This book is not
intended to give an elementary treatment of recombinant DNA technology, as
there are already a number of books in this category. The objective of writing
this book is to provide a genuine introduction in gene cloning for interested
readers with no prior knowledge in this area to learn the vocabulary and acquire
some proficiency in reading and speaking the “language”.
In the process of writing this book, the author was continuously con-
fronted with how to present the language of a complex field in a simple and
accessible manner. I have chosen to devote Part I of this book to outlining some
basic concepts of biology in a straightforward and accessible manner. My inten-
tion is to highlight only the essentials that are most relevant to understanding
gene cloning. For those who want to pursue a thorough review of genetics or
molecular biology, there are many excellent references available. Part II of the
book describes cloning techniques and approaches used in microbial, plant, as
well as mammalian systems. I believe that a discussion beyond microbes is a
prerequisite to a better comprehension of the language and the practical uses of
gene cloning. Part III describes selected applications in agriculture and food
science, and in medicine and related areas. I have taken the approach to first
introduce the background information for each application, followed by an
example of cloning strategies published in the literature. The inclusion of pub-
lications is an efficient way to demonstrate how gene cloning is conducted, and
relate it to the concepts developed in Parts I and II. Moreover, it enables the
readers to “see” the coherent theme underlining the principles and techniques of
gene cloning. Consistent with its introductory nature, the text is extensively
illustrated and the contents are developed in a logical sequence. Each chapter is
supplemented with a list of review questions as a study aid.
xi
xii Preface to the First Edition
I hope that this book will succeed in conveying not only the wonderful
language of gene cloning, but also a sense of relevance of this science in our
everyday lives. Finally, I acknowledge the contributions of my teachers and col-
leagues, especially Professor Carl A. Batt (Cornell University) and Professor
Robert E. Feeney (UC Davis), to my pursuing interest in biological molecules
and processes. Special thanks are due to Dr. Eleanor S. Reimer (Chapman &
Hall) who has been very supportive in making this book a reality.
Contents
Preface to the Third Edition vii
Preface to the Second Edition ix
Preface to the First Edition xi
Part One. Fundamentals of Genetic Processes
1 Introductory Concepts 3
1.1 What Is DNA and What Is a Gene?�� 3

1.2 What Is Gene Cloning?�� 4
1.3 Cell Organizations�� 5
1.4 Heredity Factors and Traits�� 6
1.5 Mitosis and Meiosis�� 8
1.6 Relating Genes to Inherited Traits�� 9
1.7 Why Gene Cloning? �� 10
2 Structures of Nucleic Acids 13

2.1 5′-P and 3′-OH Ends�� 13
2.2 Purine and Pyrimidine Bases�� 14
2.3 Complementary Base Pairing �� 15
2.4 Writing a DNA Molecule �� 16
2.5 Describing DNA Sizes�� 17
2.6 Denaturation and Renaturation �� 17
2.7 Ribonucleic Acid�� 18
3 Structures of Proteins 21
3.1 Amino Acids �� 21
3.2 The Peptide Bond �� 22
3.3 Structural Organization�� 24
3.4 Posttranslational Modification�� 25
3.5 Enzymes�� 26
xiii
xiv Contents
4 The Genetic Process 29

4.1 From Genes to Proteins�� 29
4.2 Transcription�� 29
4.3 Translation�� 30
4.4 The Genetic Code �� 31
4.5 Why Present a Sequence Using the Coding Strand?�� 32
4.6 The Reading Frame�� 33
4.7 DNA Replication�� 35
4.8 The Replicon and Replication Origin �� 36
4.9 Relating Replication to Gene Cloning�� 37
5 Organization of Genes 39
5.1 The Lactose Operon�� 39
5.2 Control of Transcription �� 40
5.2.1 Where Are the Transcription Start Site
and Termination Site? �� 40
5.2.2 When Does Transcription Start or Stop?�� 42
5.3 Control of Translation�� 44
5.3.1 Ribosome Binding Site and Start Codon�� 44
5.3.2 Translation Termination Site �� 44
5.4 The Tryptophan Operon�� 44
5.4.1 Co-repressor�� 45
5.4.2 Attenuation�� 45
5.4.3 Hybrid Promoters�� 47
5.5 The Control System in Eukaryotic Cells�� 47
5.5.1 Transcriptional Control �� 48
5.5.2 Introns and Exons�� 48
5.5.3 Capping and Tailing�� 49
5.5.4 Ribosome Binding Sequence�� 50
5.5.5 Monocistronic and Polycistronic�� 50
6 Reading the Nucleotide Sequence of a Gene 53

6.1 The E. coli dut Gene �� 53
6.2 The Human bgn Gene�� 55
6.2.1 Reading the Genomic Sequence�� 59
6.2.2 Reading the cDNA Sequence�� 60
Part Two. Techniques and Strategies of Gene Cloning
7 Enzymes Used in Cloning 67

7.1 Restriction Enzymes �� 67
7.2 Ligase�� 68
7.3 DNA Polymerases�� 68
7.3.1 E. coli DNA Polymerase I�� 69
Contents xv
7.3.2 Bacteriophage T4 and T7 Polymerase�� 71

7.3.3 Reverse Transcriptase�� 72
7.4 Phosphatase and Kinase�� 72
8 Techniques Used in Cloning 75

8.1 DNA Isolation�� 75
8.2 Gel Electrophoresis�� 75
8.2.1 Agarose Gel Electrophoresis�� 76
8.2.2 Polyacrylamide Gel Electrophoresis �� 76
8.3 Western Blot �� 78
8.4 Southern Transfer�� 78
8.5 Colony Blot�� 78
8.6 Hybridization�� 80
8.7 Colony PCR�� 82
8.8 Immunological Techniques�� 82
8.9 DNA Sequencing�� 84
8.10 Polymerase Chain Reaction�� 87
8.11 Site-Directed Mutagenesis�� 88
8.12 Non-radioactive Detection Methods �� 91
9 Cloning Vectors for Introducing Genes into Host Cells 93

9.1 Vectors for Bacterial Cells�� 93
9.1.1 Plasmid Vectors �� 93
9.1.2 Bacteriophage Vectors�� 99
9.1.3 Cosmids�� 102
9.1.4 Phagemids �� 103
9.2 Yeast Cloning Vectors �� 104
9.2.1 The 2 μ Circle�� 104
9.2.2 The Pichia pastoris Expression Vectors�� 106
9.3 Vectors for Plant Cells�� 106
9.3.1 Binary Vector System�� 107
9.3.2 Cointegrative Vector System �� 109
9.3.3 Genetic Markers�� 109
9.3.4 Plant Specific Promoters �� 112
9.4 Vectors for Mammalian Cells �� 112
9.4.1 SV40 Viral Vectors�� 113
9.4.2 Direct DNA Transfer�� 114
9.4.3 Insect Baculovirus�� 115
9.4.4 Retrovirus�� 119
10 Gene-Vector Construction 123
10.1 Cloning or Expression�� 123
10.2 The Basic Components �� 123
10.2.1 Expression Vectors�� 124
10.3 Reading a Vector Map�� 125
xvi Contents
10.4 The Cloning/Expression Region�� 125

10.5 The Gene Must Ligate in Frame with the Vector
for Expression�� 127
10.6 Linkers and Adapters for Introducing Restriction Sites �� 128
11 Transformation 131
11.1 Calcium Salt Treatment�� 131
11.2 Electroporation �� 132
11.3 Agrobacterium Infection�� 132
11.4 The Biolistic Process�� 132
11.5 Viral Transfection �� 133
11.6 Microinjection�� 133
11.7 Nuclear Transfer �� 134
11.8 Cell-Free Expression�� 134
12 Isolating Genes for Cloning 137

12.1 The Genomic Library �� 137
12.2 The cDNA Library�� 138
12.3 Choosing the Right Cell Types for mRNA Isolation�� 140
Part Three. Impact of Gene Cloning: Applications in Agriculture
13 Improving Tomato Quality by Antisense RNA 143

13.1 Antisense RNA �� 143
13.2 A Strategy for Engineering Tomatoes
with Antisense RNA �� 145
14 Transgenic Crops Engineered with Insecticidal Activity 149

14.1 Bacillus thuringiensis Toxins�� 149
14.2 Cloning of the cry Gene into Cotton Plants�� 150
14.2.1 Modifying the cry Gene�� 150
14.2.2 The Intermediate Vector�� 150
14.2.3 Transformation by Agrobacterium�� 150
15 Transgenic Crops Conferred with Herbicide Resistance 153

15.1 Glyphosate�� 153
15.2 Cloning of the aroA gene�� 155
16 Growth Enhancement in Transgenic Fish 157

16.1 Gene Transfer in Fish�� 157
16.2 Cloning Salmons with a Chimeric Growth
Hormone Gene�� 158
Contents xvii
Part Four. Impact of Gene Cloning: Applications

in Medicine and Related Areas
17 Microbial Production of Recombinant Human Insulin 163

17.1 Structure and Action of Insulin�� 163
17.2 Cloning Human Insulin Gene �� 164
18 Finding Disease-Causing Genes 167

18.1 Genetic Linkage�� 167
18.1.1 Frequency of Recombination�� 168
18.1.2 Genetic Markers�� 169
18.2 Positional Cloning�� 169
18.2.1 Chromosome Walking�� 170
18.2.2 Chromosome Jumping�� 171
18.2.3 Yeast Artificial Chromosome�� 171
18.3 Exon Amplification�� 172
18.4 Isolation of the Mouse Obese Gene�� 173
18.5 Exome Sequencing �� 173
18.5.1 Targeted Enrichment by Sequence Capture�� 174
18.5.2 Disease Gene Identification�� 175
19 Human Gene Therapy 177

19.1 Physical and Chemical Methods�� 177
19.2 Biological Methods�� 179
19.2.1 Life Cycle of Retroviruses�� 179
19.2.2 Construction of a Safe Retrovirus Vector�� 179
19.2.3 Gene Treatment of Severe Combined Immune
Deficiency �� 180
19.3 Adeno-Associated Virus �� 181
19.3.1 Life Cycle of Adeno-Associated Virus �� 182
19.3.2 Recombinant Adeno-Associated Virus�� 182
19.3.3 Recombinant Adeno-Associated
Virus-Mediated Gene Treatment for Leber’s
Congenital Amaurosis Type 2 �� 184
19.4 Therapeutic Vaccines�� 184
19.4.1 Construction of DNA Vaccines �� 185
19.4.2 Delivery of DNA Vaccines�� 185
20 Gene Targeting and Genome Editing 187

20.1 Recombination�� 187
20.2 Replacement Targeting Vectors�� 188
20.3 Gene Targeting Without Selectable Markers�� 189
20.3.1 The PCR Method�� 190
20.3.2 The Double-Hit Method�� 190
20.3.3 The Cre/loxP Recombination�� 191
xviii Contents
20.4 Gene Targeting for Xenotransplants �� 192

20.5 Engineered Nucleases: ZFN, TALEN, CRISPR�� 193
20.5.1 Zinc-Finger Nucleases�� 194
20.5.2 Transcription Activator-Like Effector
Nucleases�� 194
20.5.3 The CRISPR/Cas System�� 195
20.5.4 Nonhomologous End Joining
and Homology-Directed Repair�� 196
20.5.5 Expressing Engineered Nucleases
in Target Cells �� 196
21 DNA Typing 199
21.1 Variable Number Tandem Repeats�� 199
21.2 Polymorphism Analysis Using VNTR Markers �� 200
21.3 Single-Locus and Multi-locus Probes�� 201
21.4 Paternity Case Analysis�� 201
21.5 Short Tandem Repeat Markers�� 202
21.5.1 The Combined DNA Index System�� 204
21.6 Mitochondrial DNA Sequence Analysis�� 205
22 Transpharmers: Bioreactors for Pharmaceutical Products 209

22.1 General Procedure for Production of Transgenic
Animals�� 210
22.2 Transgenic Sheep for α1-Antitrypsin�� 210
23 Animal Cloning 213
23.1 Cell Differentiation �� 213
23.2 Nuclear Transfer �� 214
23.3 The Cloning of Dolly�� 215
23.4 Gene Transfer for Farm Animals�� 216
24 Whole Genome and Next Generation Sequencing 219

24.1 Genetic Maps�� 219
24.1.1 DNA Markers�� 220
24.1.2 Pedigree Analysis�� 220
24.2 Physical Maps�� 221
24.2.1 Sequence Tagged Sites�� 221
24.2.2 Radiation Hybridization�� 221
24.2.3 Clone Libraries�� 222
24.2.4 The Bacterial Artificial Chromosome Vector�� 223
24.3 Comprehensive Integrated Maps�� 224
Contents xix
24.4 Strategies For Genome Sequencing�� 224

24.4.1 Hierarchical Shotgun Sequencing �� 224
24.4.2 Whole-Genome Shotgun Sequencing �� 226
24.5 Next Generation Sequencing of Whole Genomes�� 226
24.5.1 The Basic Scheme of NGS�� 227
Suggested Readings 231
Index 245
Part One
Fundamentals of Genetic
Processes
chapter 1
Introductory Concepts
The building blocks of all forms of life are cells. Simple organisms
such as bacteria exist as single cells. Plants and animals are composed of many
cell types, each organized into tissues and organs of specific functions. The
determinants of genetic traits of living organisms are contained within the
nucleus of each cell, in the form of a type of nucleic acids, called deoxyribo-
nucleic acid (DNA). The genetic information in DNA is used for the synthesis
of proteins unique to a cell. The ability of cells to express the information coded
by DNA in the form of protein molecules is achieved by a two-stage process of
transcription and translation.
Transcription Translation
DNA _____________________> _____________________> Protein
1.1 What Is DNA and What Is a Gene?
A DNA molecule contains numerous discrete pieces of information,

each coding for the structure of a particular protein. Each piece of the informa-
tion that specifies a protein corresponds to only a very small segment of the
DNA molecule. Bacteriophage λ, a virus that infects bacteria, contains all its 60
genes in a single DNA molecule. In humans, there are about 20,000 genes orga-
nized in 46 chromosomes, complex structures of DNA molecules associated
with proteins.
When, how, and where the synthesis of each protein occurs is precisely
controlled. Biological systems are optimized for efficiency; proteins are made
only when needed. This means that transcription and translation of a gene in the
production of a protein are highly regulated by a number of control elements,
many of which are also proteins. These regulatory proteins are in turn coded by
a set of genes.
© Springer International Publishing AG, part of Springer Nature 2018 3

D. W. S. Wong, The ABCs of Gene Cloning,
https://doi.org/10.1007/978-3-319-77982-9_1
4 The ABCs of Gene Cloning
It is therefore more appropriate to define a gene as a functional unit. A

gene is a combination of DNA segments that contain all the information neces-
sary for its expression, leading to the production of a protein. A gene defined in
this context would include (1) the structural gene sequence that encodes the
protein, and (2) sequences that are involved in the regulatory function of the
process.
1.2 What Is Gene Cloning?
Gene cloning is the process of introducing a foreign DNA (or gene)

into a host (bacterial, plant, or animal) cell. In order to accomplish this, the gene
is usually inserted into a vector (a small piece of DNA) to form a recombinant
DNA molecule. The vector acts as a vehicle for introducing the gene into the
host cell and for directing the proper replication (DNA -> DNA) and expression
(DNA -> protein) of the gene (Fig. 1.1).
The process by which the gene-containing vector is introduced into a
host cell is called “transformation”. The host cell now harboring the foreign
gene is a “transformed” cell or a “transformant” .
The host cell carrying the gene-containing vector produces progeny all
of which contain the inserted gene. These identical cells are called “clones”.
In the transformed host cell and its clones, the inserted gene is tran-
scribed and translated into proteins. The gene is therefore “expressed”, with the
gene product being a protein. The process is called “expression”.
Fig. 1.1. General scheme of gene cloning

Introductory Concepts 5
1.3 Cell Organizations
Let us focus the attention for a moment on the organization and the
general structural features of a cell, knowledge of which is required for com-
manding the language of gene cloning. Cells exist in one of two distinct types
of arrangements (Fig. 1.2). In a simple cell type, there are no separate compart-
ments for genetic materials and other internal structures.
Fig. 1.2. Drawing of cells showing details of organelles

Organisms with this type of cellular organization are referred to as pro-

karyotes. The genetic materials of prokaryotes, such as bacteria, are present in
a single circular DNA in a clear region called nucleoid that can be observed
microscopically. Some bacteria also contain small circular DNA molecules
called plasmids. (Plasmids are the DNA used to construct vectors in gene clon-
ing. See Sect. 9.1.) The rest of the cell interior is the cytoplasm, which contains
numerous minute spherical structures called ribosomes – the sites for protein
synthesis. Defined structures like ribosomes, are called organelles. The rest
(fluid portion) of the cytoplasm is the cytosol, a solution of chemical constitu-
ents that maintain various functions of the cell. All the intracellular materials are
enclosed by a plasma membrane, a bilayer of phospholipids in which various
proteins are embedded. In addition, some bacterial cells contain an outer layer
of peptidoglycan (a polymer of amino-sugars) and a capsule (a slimy layer of
polysaccharides).
In contrast, a vast majority of living species including animals, plants,
and fungi, have cells that contain genetic materials in a membrane-bound
nucleus, separated from other internal compartments which are also surrounded
by membranes. Organisms with this type of cell organization are referred to as
eukaryotes. The number and the complexity of organelles in eukaryotic cells far
exceed those in bacteria (Fig. 1.2). In animal cells, the organelles and constitu-
ents are bound by a plasma membrane. In plants and fungi, there is an additional
outer cell wall that is comprised primarily of cellulose. (In plant and fungal
cells, the cell wall needs to be removed before a foreign DNA can be introduced
into the cell in some cases as described in Sect. 11.1).
1.4 Heredity Factors and Traits
In a eukaryotic nucleus, DNA exists as complexes with proteins to

form a structure called chromatin (Fig. 1.3). During cell division, the fibrous-
like chromatin condenses to form a precise number of well-defined structures
called chromosomes, which can be seen under a microscope.
Chromosomes are grouped in pairs by similarities in shape and length
as well as genetic composition. The number of chromosome pairs varies in dif-
ferent species. For example, carrots have 9 pairs of chromosomes, humans have
23 pairs, and so on. The two similar chromosomes in a pair are described as
homologous, containing genetic materials that control the same inherited traits.
If a heredity factor (gene) that determines a specific inherited trait is located in
one chromosome, it is also found at the same location (locus) on the homolo-
gous chromosome. The two copies of a gene that are found in the same loci in a
homologous chromosome pair are determinants of the same hereditary trait, but
may exist in various forms (alleles). In simple terms, dominant and recessive
alleles exist for each gene.
Fig. 1.3. Structure of cellular chromosome
In a homologous chromosome pair, the two copies of a gene can exist

in three types of combinations: 2 dominant alleles, 1 dominant and 1 recessive,
or 2 recessives. Dominant alleles are designated by capital letters, and recessive
alleles by the same letter but in lower case. For example, the shape of a pea seed
is determined by the presence of the R gene. The dominant form of the gene is
“R”, and the recessive form of the gene is designated as “r”. The homologous
combination of the alleles can be one of the following: (1) RR (both dominant),
(2) Rr (one dominant, one recessive) or (3) rr (both recessive). This genetic
makeup of a heredity factor is called the genotype. A dominant allele is the form
of a gene that is always expressed, while a recessive allele is suppressed in the
presence of a dominant allele. Hence, in the case of the genotypes RR and Rr, the
pea seeds acquire a round shape, and a genotype of rr will give a wrinkled seed.
The observed appearance from the expression of a genotype is its phenotype.
In the example, a pea plant with a genotype of RR or Rr has a pheno-
type of round shape seeds. When two alleles of a gene are the same (such as RR
or rr), they are called homozygous (dominant or recessive). If the two alleles are
different (such as Rr), they are heterozygous. The genotypes and phenotypes of
the offspring from breeding between, for example, two pea plants having geno-
types of Rr (heterozygous) and rr (homozygous recessive), can be tracked by
the use of a Punnett square (Fig. 1.4a). The offspring in the first generation will
have genotypes of Rr and rr in a 1:1 ratio, and phenotypes of round seed and
wrinkled seed, respectively.
Fig. 1.4. Cross between (a) Rr and rr pea plants, and (b) carrier female
and normal male
The example of round/wrinkled shape of pea seeds is typical of one

gene controlling a single trait. The situation is more complex in most cases,
because many traits are determined by polygenes. Eye color, for example, is
controlled by the presence of several genes. In some cases, a gene may exist in
more than two allelic forms. Human ABO blood types are controlled by a gene
with 3 alleles – IA and IB are codominant, and Io is recessive. Additional varia-
tions are introduced by a phenomenon called crossing over (or recombination)
in which a genetic segment of one chromosome is exchanged with the corre-
sponding segment of the homologous chromosome during meiosis (a cell divi-
sion process, see Sects. 1.5 and 18.1).
A further complication arises from sex-linked traits. Humans have 23
pairs of chromosomes. Chromosome pairs 1 to 22 are homologous pairs, and
the last pair contains sex chromosomes. Male has XY pair and female has XX
chromosomes. The genes carried by the Y chromosome dictate the development
of a male; the lack of the Y chromosome results in a female. A sex-linked gene
is a gene located on a sex chromosome. Most known human sex-linked genes
are located on the X chromosome, and thus are referred to as X-linked. An
example of a sex-linked trait is color blindness, which is caused by a recessive
allele on the X chromosome (Fig. 1.4b). If a carrier female is married to a nor-
mal male, the children will have the following genotypes and phenotype- Sons:
XY (color blind) and XY (normal), and daughters: XX (normal, carrier) and XX
(normal, non-carrier).
1.5 Mitosis and Meiosis
The presence of homologous chromosome pairs is the result of sexual

reproduction. One member of each chromosome pair is inherited from each par-
ent. In human and other higher organisms, autosomal cells (all cells except the
germ cells, sperms and eggs) contain a complete set of homologous
chromosomes, one of each pair from one parent. These cells are called diploid
cells (2n). Germ cells contain only one homolog of each chromosome pair, and
are referred to as haploid (n).
A fundamental characteristic of cells is their ability to reproduce
themselves by cell division – a process of duplication in which two new (daugh-
ter) cells arise from the division of an existing (parent) cell. Bacterial cells
employ cell division as a means of asexual reproduction, producing daughter
cells by binary fission. The chromosome in a parent cell is duplicated, and
separated so that each of the two daughter cells acquires the same chromosome
as the parent cell.
In eukaryotes, the process is not as straightforward. Two types of cell
division, mitosis and meiosis, can be identified. In mitosis, each chromosome is
copied into duplicates (called chromatids) that are separated and partitioned
into two daughter cells. Therefore, each of the two daughter cells receives an
exact copy of the genetic information possessed by the parent cell (Fig. 1.5).
Mitosis permits new cells to replace old cells, a process essential for growth and
maintenance. In meiosis, the two chromatids of each chromosome stay attached,
and the chromosome pairs are separated instead, resulting in each daughter cell
carrying half of the number of chromosomes of the parent cell (Fig. 1.5). Note
that at this stage, each chromosome in the daughter cells consists of 2 chroma-
tids. In a second step of division, the chromatids split, resulting in 4 daughter
cells each containing a haploid number of chromosomes, i.e. only one member
of each homologous chromosome pair. Meiosis is the process by which germ
cells are produced. After fertilization of an egg with a sperm, the embryo has
complete pairs of homologous chromosomes.
1.6 Relating Genes to Inherited Traits
The preceding discussions on dominant and recessive forms, and geno-

types and phenotypes, can be interpreted at the molecular level by relating them
to how genes determine inherited traits. In simple terms, a gene can exist in a
functional form, so that it is expressed through transcription and translation to
yield a gene product (a specific protein) that exhibits its normal function.
However, a gene can also be non-functional due to a mutation, for example,
resulting in either the absence of a gene product, or a gene product that does not
function properly. Therefore, a homozygous dominant genotype, such as AA,
means that both alleles in the chromosome pair are functional. A genotype of Aa
will still have one functional copy of the gene that permits the synthesis of the
functional protein. A homozygous recessive (aa) individual does not produce
the gene product or produce a nonfunctional gene product. A gene controls an
inherited trait through its expression, in that the gene product determines the
associated inherited characteristic. Genes with multiple alleles can be explained
Fig. 1.5. Schematic comparison between mitosis and meiosis
by the difference in the efficiencies of the functions of the gene products.

Another explanation is that one copy of the gene produces a lower amount of the
gene product than the corresponding normal (functional) gene.
An example can be drawn from the genetic disorder of obesity in mice.
Obese (ob) is an autosomal recessive mutation in chromosome 6 of the mouse
genome. The normal gene encodes the Ob protein, which functions in a signal
pathway for the body to adjust its energy metabolism and fat accumulation (see
Sect.18.4). Mice carrying 2 mutant copies (ob/ob) of the gene develop progres-
sive obesity with increased efficiency in metabolism (i.e. increase weight gain
per calorie intake). Mice with ob/ob genotype do not produce the gene product
(Ob protein), because both copies of the ob gene are nonfunctional.
1.7 Why Gene Cloning?
The general objective of gene cloning is to manipulate protein synthesis.

There are several reasons why we want to do this.
1. To produce a protein in large quantity. Large-scale production of
therapeutic proteins has been a primary focus of biotechnology. Many proteins
of potential therapeutic values are often found in minute amounts in biological

systems. It is not economically feasible to purify these proteins from their natu-
ral sources. To circumvent this, the gene of a targeted protein is inserted into a
suitable host system that can efficiently produce the protein in large quantities.
Examples of pharmaceuticals of this type include human insulin, human growth
hormone, interferon, hepatitis B vaccine, tissue plasminogen activator, interleu-
kin-2, and erythropoietin. Another area of great interest is the development of
“transpharmers”. The gene of a pharmaceutical protein is cloned into livestock
animals, and the resulting transgenic animals can be raised for milking the
protein.
2. To manipulate biological pathways. One of the common objectives
in gene cloning is to improve crop plants and farm animals. This often involves
alteration of biological pathways either by (A) blocking the production of an
enzyme, or (B) implementing the production of an exogenous (foreign) enzyme
through the manipulation of genes. Many applications of gene cloning in agri-
culture belong to the first category. A well-known example is the inhibition of
the breakdown of structural polymers in tomato plant cell wall by blocking the
expression of the gene for the enzyme involved in the breakdown process (using
antisense technique). The engineered tomatoes, with decreased softening, can
be left to ripe on the vine, allowing full development of color and flavor. Another
example is the control of ripening by blocking the expression of the enzyme that
catalyzes the key step in the formation of the ripening hormone, ethylene.
On the other hand, new functions can be introduced into plants and
animals by introducing a foreign gene for the production of new proteins that
are previously not present in the system. The development of pest-resistant
plants has been achieved by cloning a bacterial endotoxin. Other examples
include salt-tolerant and disease-resistant crop plants. Similar strategies can be
applied to raise farm animals, with build-in resistance to particular diseases.
Animals cloned with growth hormone genes result in the enhancement of
growth rate, increased efficiency of energy conversion, and increased protein to
fat ratio. All these translate into lower cost of raising farm animals, and a lower
price for high quality meat.
A number of human genetic diseases, such as severe-combined immu-
nodeficiency (SCID), are caused by the lack of a functional protein or enzyme,
due to a single defective gene. In these cases, the defect can be corrected by the
introduction of a healthy (normal, therapeutic) gene. The augmentation enables
the patient to produce the key protein required for the normal functioning of the
biological pathway. “Naked” DNA such as plasmids containing the gene
encoding specific antigens can be used as therapeutic vaccines to stimulate
immune responses for protection against infectious diseases.
3. To change protein structure and function by manipulating its gene.
One can modify the physical and chemical properties of a protein by altering its
structure through gene manipulation. Using the tools in genetic engineering, it

is possible to probe into the fine details of how proteins function, by investigat-
ing the effects of modifying specific sites in the molecule. This technique has
generated vast information on our current knowledge on the mechanism of
important proteins and enzyme functions.
For therapeutic applications, many of the proteins are engineered to
modify the structure and activity. For example, crosslinking the variable domains
of different monoclonal antibodies by short peptide linkers can form single-
chain bispecific antibodies that are less immunogenic with enhanced tissue pen-
etration. Glycoengineering has been applied to introduce sugar moieties into
antibodies to improve solubility and increase the half-life of the protein.
Modifying the proteolytic cleavage site of coagulation factor VIII enhances its
resistance to inactivation for improved pharmacokinetic properties.
For illustration of the impact of gene cloning, some application exam-
ples are covered in Part III (for agriculture) and Part IV (for medicine and related
areas) of this book.
Review
1. Define: (A) a gene, (B) transformation, (C) a clone, (D) expression.
2. What is a vector used for?
3. List some applications of gene cloning.
4. Describe the differences in structural features between prokaryotic and eukaryotic
cells.
5. Match by circling the correct answer in the right column.
Homozygous dominant RR, Rr, rr
Homozygous recessive RR, Rr, rr
Heterozygous RR, Rr, rr
6. Tongue rolling is an autosomal recessive trait. What are the genotypes and pheno-
types of the children from a heterozygous female married to a homozygous dominant
male?
7. Hemophilia is a sex-linked trait. Describe the genotypes and phenotypes of the sons
and daughters from a marriage between a normal male and a carrier female.
8. Identify the differences between mitosis and meiosis.
Mitosis Meiosis
(A) Number of daughter cells
(B) Haploid or diploid
(C) One or two divisions
(D) Germ cells or somatic cells
9. Why is it that a dominant allele corresponds to a functional gene? Why is it recessive

if a gene is nonfunctional?
chapter 2
Structures of Nucleic Acids
What is the chemical structure of a deoxyribonucleic acid (DNA) mol-

ecule? DNA is a polymer of deoxyribonucleotides. All nucleic acids consist of
nucleotides as building units. A nucleotide has three components: sugar, base,
and a phosphate group. (The combination of a sugar and a base is a nucleoside.)
In the case of DNA, the nucleotide is known as deoxyribonucleotide, because
the sugar in this case is deoxyribose. The base is either a purine (adenine or
guanine) or a pyrimidine (thymine or cytosine) (Figs. 2.1 and 2.3). Another type
of nucleic acid is ribonucleic acid (RNA), a polymer of ribonucleotides also
consisting of three components – a sugar, a base and a phosphate. The sugar in
this case is a ribose, and that the base thymine is replaced by uracil (Sect. 2.7).
2.1 5′-P and 3′-OH Ends
In DNA, the hydroxyl (OH) group is attached to the carbon at the 3′

position of the deoxyribose. One of the three phosphates (P) in the phosphate
group is attached to the carbon at the 5′ position (Fig. 2.1). The OH group and
the P group in a nucleotide are called 3′-OH (3 prime hydroxyl) and 5′-P (5
prime phosphate), respectively. A nucleotide is more appropriately described as
2′-deoxynucleoside 5′-triphosphate to indicate that the OH at the 2′ position is
deoxygenated and the phosphate group is attached to the 5′ position.
A DNA molecule is formed by linking the 5′-P of one nucleotide to the
3′-OH of the neighboring nucleotide (Fig. 2.2). A DNA molecule is therefore a
polynucleotide with nucleotides linked by 3′-5′ phosphodiester bonds. The 5′-P
end contains three phosphates but in the 3′-5′ phosphodiester bonds, two of the
phosphates have been cleaved during bond formation. An important conse-
quence to a phosphodiester linkage is that DNA molecules are directional: one
end of the chain with a free phosphate group, and the other end with a free OH
group. It is important in cloning to specify the two ends of a DNA molecule:
5′-P end (or simply 5′ end) and 3′-OH end (or 3′ end).
© Springer International Publishing AG, part of Springer Nature 2018 13

D. W. S. Wong, The ABCs of Gene Cloning,
https://doi.org/10.1007/978-3-319-77982-9_2
Fig. 2.1. Chemical structure of deoxyribonucleotide
Fig. 2.2. Polynucleotide showing a 3′-5′ phosphodiester bond
2.2 Purine and Pyrimidine Bases
The deoxyriboses and phosphate groups forming the backbone of a

DNA molecule are unchanged throughout the polynucleotide chain. However,
the bases in the nucleotides vary because there are 4 bases – adenine, thymine,
guanine and cytosine, abbreviated as A, T, G and C, respectively (Fig. 2.3,
Table 2.1). A and G are purines (with double-ring structures); T and C are
pyrimidines (with single-ring structures). Consequently, there are four different
nucleotides.
Exploring the Variety of Random
Documents with Different Content
H. G. HINE
Strangely little notice, considering the artistic importance of the
subject, has been taken of the death of H. G. Hine, the eminent artist
in water-colours, vice-president of the Royal Institute, who died a
fortnight ago, aged eighty-three years. The explanation, I fear, of the
scanty comment his death has evoked, is to be sought in the fact
that the mass of that public which concerns itself with Art at all, is
occupied chiefly with such art as exhibits an easy piquancy of
treatment or an obvious interest of subject. Hine’s did neither; yet the
best-equipped critics have long done justice to the steady perfection
with which he dealt with those themes of serene weather upon ‘the
billows of the Downs,’ which—superlatively though they were
executed by him—he, with a hankering sometimes after other
compositions and other effects, declined to consider his speciality.
Yet a speciality, of course, they were: those visions of turquoise or of
opal sky, and of grey gold or of embrowned gold turf, with the long,
restful sweeps and subtle curves, the luminous shadows, the points
of light, with the shepherd and his flock on the ascending hillside,
with the ancient thorn-tree bent by the winds of many an autumn.
Singularly unlike the work of strange refinement and
unsurpassed subtlety which it was his wont to produce, was Hine
himself, with his sturdy and sailorlike personality. Yet the character of
the man was, in truth, not less admirable than the artistic finesse of
his work. He found his true path somewhat late in life. His genius
came to him almost as tardily, but then, perhaps, almost as
powerfully, as did David Cox’s. He was long past fifty when—with a
charm of composition not less certain than Copley Fielding’s, and
with the genius of a far finer and fuller colourist—he began to do
justice to the Sussex Downs, amid whose generally unconsidered
scenery it had been his excellent fortune to be born.
(Academy, 30th March 1895.)
THOMAS COLLIER
English landscape art—the practice of which he had adorned by
five-and-twenty years of noble work—sustains a profound loss in the
death of Thomas Collier. He was born in the year 1840, at Glossop,
on the Derbyshire border. He early addressed himself to the career
of a landscape painter; and it is true, no doubt, that his method was
founded upon that of David Cox, nor is it possible that he could have
set up for himself a better model of delicacy of observation, and of
decisive and economical handwork. And the medium of Collier was
—like that of David Cox—almost exclusively water-colour. His oil
paintings were few, and, like Cox’s, they were executed chiefly in his
later time. But, with him, the later time was still only middle age.
Collier died when he was fifty-one: David Cox at seventy-six. Had
David Cox left us at the age of Collier, he would hardly have been
remembered to-day, and could have been an example to no one.
Collier passed through no such prolonged period of preparation for
mastery. He was already a master in his early manhood. His work
cannot well be divided into periods: freedom of manner, largeness of
vision and touch, belonged to him almost from the first. To the quite
superficial observer of his drawings, it appeared that he painted only
two or three subjects, and these on the same grey day. But to the
real student of his work, the richness and variety of his resource is
revealed. He observed and recorded differences of weather and light
which escape all casual and all untrained notice; and if he was
among the simplest and most vigorous, he was also among the most
poetic recorders of English countryside and homestead—of farm,
and coast, and moor. His work, exhibited in France, obtained for him
the decoration of a chevalier of the Legion of Honour, and here in
England he was one of the most distinguished members of the Royal
Institute of Painters in Water-Colours. But it is doubtful whether the
opportunities afforded to the large public for seeing his work were
frequent enough to secure him that degree of actual popularity which
was his due; and it is at all events certain that when the cabinet of
sketches which he showed occasionally to his friends shall come to
be known more widely, Collier will be accorded, without cavil or
questioning, a lasting place among the Masters.
(Academy, 23rd May 1891.)

LORD LEIGHTON
By the death of Lord Leighton, the Royal Academy loses a great
President and England a many-sided artist, who was certainly not far
removed from being a great painter. It was more, perhaps, by the
combination of so many various qualities of character and talent than
by the firm possession of one especial vein of genius, that ‘our dear
President, our admirable Leighton’—to use the words most fittingly
applied to him by Sir John Millais—had come, of recent years at
least, to be distinguished and known. The painter’s and designer’s
art, evidenced in his youth, about forty years ago, by the ‘Procession
of Cimabue,’ had not only never fallen into disuse, but had never
come to occupy, in his mind, a secondary or comparatively
unregarded place. But, along with the well-maintained devotion to
the craft to which he had first vowed his affections a full generation
ago, there had sprung up, partly of necessity and partly by reason of
Lord Leighton’s exceptional temperament, many interests, exclusive
of merely official duties, which occupied time and thought—so much
so that if he had not added to the tastes of an artist the habits and
qualifications of a great man of affairs, it would have been impossible
for him to have successfully crowded into his life all the pursuits that
engrossed it. It is easy for the ‘admirable Crichton,’ in these modern
times, to degenerate into the Mr. Brook of Middlemarch—the not
unamiable dilettante who was pretty certain to have once ‘taken up’
everything, and was pretty certain also to have dropped it. But Lord
Leighton, great as was the diversity of his interests, was absolutely
systematic and thoroughgoing; and, outside his especial art (in which
his place, whatever may have been his deficiencies, was peculiar
and unquestioned), he not only practised but excelled.
Leighton was linguist, student, antiquary, man of fashion,
administrator, even philanthropist. His oratory was an
accomplishment; albeit, in its addiction to ingenious ornament, his
style was not quite of our period. His tact in dealing with men and
with affairs was almost faultless. His opinions were decided, and he
never concealed them; yet, in uttering them, he hardly ever gave
offence—never, indeed, to the reasonable. When all these things are
remembered, and when there is added to them the recollection of a
presence elegant and stately, and of a manner which, though it could
well keep intruders at a distance, had singular and winning charm for
the many whom it was intended to please, it will be fully realised
what a difficult and heavy honour awaits Lord Leighton’s successor
in his great function—that of President of the Royal Academy, and
official representative of English Art. The Academy contains several
painters of genius; several amiable and distinguished men of the
world; but as those who can look back the furthest declare that no
past President of whom they had any knowledge ever equalled Lord
Leighton, it may well be doubted whether a future President is likely
to equal him.
So much by way of rough indication of the character of the man,
and of the public man. A further explanation of his individuality must,
of course, be discovered in his Art; and even a cursory survey of it—
and of the creations which were the events of his life—will disclose
something of his strength, and something, too, of his weakness. The
son of a physician whose life was extended to a most ripe old age,
and grandson of Sir James Leighton, also a doctor—long resident at
the Court of St. Petersburg—Frederic Leighton was born at
Scarborough, on the 3rd December 1830. A Yorkshireman in fact—
like William Etty, and another remarkable artist of a later generation,
Thomas Collier—no one could have been less of a Yorkshireman in
character than was the late President. To what is understood or
conjectured to have been a Jewish strain in his blood are possibly to
be attributed his profoundly artistic inclinations, which were
manifested very early, and which, as the public knows, dominated
the whole of his career. It is recorded that young Leighton received
drawing lessons in Rome as long ago as the year 1842; and not two
years afterwards he entered as a student at the Academy of Berlin.
With Rome, perhaps, began that long series of Wanderjahre which
made him so cosmopolitan an artist and so many-sided a man. He
had some general education at Frankfort; then, after a removal to
Florence, where the American sculptor, Hiram Powers, was
consulted with a view to an opinion on his ability, and prophesied
that the boy ‘could become as eminent as he pleased,’ young
Leighton’s father withdrew his long-standing objections to the
adoption of painting as a profession; and the new decision was
followed by a sojourn in Brussels and a longer stay in Paris. In Paris
the youth attended a life-school, and copied at the Louvre. Next we
hear of him at Vienna, where he was a pupil of Steinle, himself a
pupil of Overbeck. Of Overbeck’s religious unction, Leighton had
never a perceptible share. Something he no doubt owed to the
leaders of the German Renaissance of Painting; but amongst these,
more, it may be, to Cornelius than Overbeck. After his sojourn in
Vienna, he was back again in Rome—these early and most
prolonged wanderings are worthy of chronicle, because they had so
much to do with the formation of the characteristics of the artist—and
it was from Rome that he sent to the Royal Academy Exhibition of
1855 a picture which made no bid for immediately popular effect,
which was nothing, moreover, of a ‘pot-boiler,’ and which made no
concession to ordinary bourgeois liking. It was the canvas in which is
depicted, with something of reticence and grace, and with a very
learned draughtsmanship, the procession which passed through the
streets of Florence, on its way to Santa Maria Novella, when
Cimabue’s picture of the Madonna was carried in the midst, and
honour and peculiar recognition—in which a whole city joined—were
bestowed upon its painter. Elegant as the picture was, it did not lack
favour; a certain relative warmth, a certain romantic spirit, the
presentation of the ideal, it may be, in more homely form, pleased a
generation familiar with Dyce, Maclise, and Cope; and the picture, as
it happened, had an immediate success.
Paris was Leighton’s next halting-place, and now, an artist rising
above the horizon, he was no longer likely to seek direct instruction
from any one of the painters who were there at work; but he was
associated with, and was to some extent influenced by, men like Ary
Scheffer (whose ‘Augustine and Monica’ was long appreciated in
England) and Robert Fleury. He contributed almost without
intermission, for the next eight or nine years, to the Royal Academy,
and it was in 1864, when he was represented by an ‘Orpheus and
Eurydice,’ that he was elected to the Associateship—becoming in
1869 a full member. The year of his election to the Associateship
was likewise the year of the exhibition of his charming and seductive
invention, ‘Golden Hours.’ To the painter of mediæval or
Renaissance history, and of themes avowedly classic, there was
vouchsafed the expression of the romantic and the unquestionably
poetic, and it is, no doubt, to the certain element of poetry that is in
Lord Leighton’s work—far more, at all events, than to its austerer
qualities of design, which never had any popularity at all, and which,
even amongst painters, have gone terribly out of fashion—that is to
be attributed part of the great favour which his art has enjoyed. In
1869 was shown ‘Electra at the Tomb of Agamemnon,’ and in 1876
the second great processional, ‘The Daphnephoria.’ Two years later
the ‘Arts of War’—not the least dignified and decorative of modern
frescoes—was finished for South Kensington, where was already its
companion, ‘The Industrial Arts of Peace,’ completed in 1873;
another mural painting, that of ‘The Wise and Foolish Virgins,’
having, at an earlier date, been placed in the chancel of a fortunate
parish church in Hampshire. The year of the completion of ‘The Arts
of War’ was that of Lord Leighton’s election to the Presidency of the
Academy, which he obtained, it will be remembered, in direct
succession to Sir Francis Grant, with whose courtly qualities, and
with whose large and manly sympathies, he combined a width of
artistic outlook, a refinement of artistic expression, which had
scarcely perhaps belonged to any President of the Academy since
the days of its first leader, Sir Joshua Reynolds.
President, and knighted in consequence of that distinction in
1878, Leighton was given a baronetcy in 1886. In the interval he had
not only proved beyond dispute his fitness for the responsibilities of
the official position, which he filled, but—to mention only some of the
most memorable of many works—had completed his own portrait for
the Uffizi, had wrought the really grave and impressive canvas of
Elisha raising the son of the Shunamite widow, and had, in his
peculiar fashion, effected an alliance between luxury in colour and
sculpturesque arrangement of ‘line’ in the great ‘Cymon and
Iphigenia.’ In actual Sculpture, too—sharing the ambition of the men
of the Renaissance for a triumph in various mediums—he had
produced ‘The Sluggard.’ It was extraordinarily clever, but perhaps
its qualities were less truly sculptural than was some of his design
executed in the older and more familiar material. Yet, if this particular
work did not possess to the full all the great qualities that might have
been expected in it, the order of Lord Leighton’s talent was one,
nevertheless, which empowered him to succeed thoroughly in
Sculpture, sooner or later; for, in Sculpture, while there was room for
the generally unimpeded play of his own skill in design, there might
have been a relief found from the exercise of his art in a path in
which success to him was more uncertain and capricious—the path
of colour.
It is too early, of course, to attempt to settle definitely the place of
Leighton in English Art; but it is certain that his influence, whether as
President or painter, tended to the extension of its vistas. An
upholder of the Classic—never, with all his range, much in love with
Realism—he was yet nothing whatever of a partisan, and—it may be
mentioned as a characteristic detail of him in his daily ways—he was
accustomed from time to time to purchase clever little drawings
(sometimes the very last one would have thought he would care for)
by artists who esteemed him as a President, but who regarded him
very lightly as a practitioner of their own craft. Lord Leighton was
perfectly aware that several circumstances limited—especially of late
years—the appreciation of his work. He was not altogether
insensible of its real defects—at all events, of peculiarities which
were defects upon occasion. He knew that his ‘brush-work’ was not
absolutely ‘modern.’ He must have allowed that, now and again,
when it was by no means one of his aims to seek it, the texture of his
flesh was porcelain-like, and thus mainly conventional. He was,
confessedly, not greatly occupied with ‘values’ of colour, with the
relation of part to part. He was at one—perhaps more than they
knew it—with many of our newest artists in demanding a decorative
quality; only the decorative quality of his choice was not always—
was, indeed very seldom—that of theirs. A successful pattern of
colour they could understand the virtue of. The Japanese, or Mr.
Whistler, had taught it them. But a successful pattern of line, they
were less capable of appreciating. They, for example, or some of
them, execrated Bouguereau, and resented in some degree the
hospitality prominently offered to that distinguished Frenchman on
the walls of the Academy. Lord Leighton, on the other hand, was,
very possibly, not fully alive to Bouguereau’s vices or failings—to his
mere smoothness, softness, not infrequent vapidness of human
expression. But he valued justly Bouguereau’s possession of the
best Academic graces, of faultless composition and subtle
draughtsmanship. For these things—these best Academic graces—
he himself strove. These, too, he generally, though not always,
attained.
In regard to this particular matter, there were times when
Leighton knew himself to be a vox clamantis in deserto. But he had
his mission. It is an immense tribute to him to recognise that any one
caring, as he undoubtedly cared, to be acceptable amongst his
fellows—amongst the younger men, even, who were some day to
succeed him—should yet have been so true to his particular
message. But Lord Leighton had an admirable courage as well as a
great patience and an untiring diligence. And there were times,
fortunately, when it was brought home to him beyond cavil, that
some educated appreciation existed of his own especial artistic
qualities, as well as of those human virtues which made him, in
many ways, so estimable a man, and so fitting a leader of men.
(Standard, 27th January 1896.)

SIR JOHN MILLAIS
For the second time within a few months the Royal Academy has
lost its chief, while English Painting is deprived of its most popular
representative, and contemporary English Art of one who was long
its most vigorous and most varied personality. Born at Southampton
in 1829, the ‘son of John William Millais, Esquire, by Mary, daughter
of Richard Evemy, Esquire’—as the official biographies relate—
Millais was really the descendant of a Jersey family of long standing;
but in character, personal and professional, he was typically English.
It is partly by reason of the fact that, as a man and as an artist,
Millais summed up some, perhaps, of the defects, many certainly of
the great qualities, of our English race, that his popularity amongst
all personal associates, and amongst the spectators of his decisive,
strenuous, and eager work, was won so early, and has been so
firmly held.
The man himself, during forty years or thereabouts of active
adult life—the artist during forty years of scarcely relaxed endeavour
—has been in thought, in conduct, in taste, and in production, pre-
eminently healthy. Millais, in the generation and a half of his active
life—for he began young—had seen fashions good and bad, foolish
and reasonable, rise and pass away; but, save by the influences of
his quite early days, the days of the Pre-Raphaelites, he has been
practically unaffected. He has developed in the direction proper to
himself. As time has passed, he and his sympathies have broadened
and modified, and if we miss in much of the later work the intense
and concentrated poetry of the earlier, that later work has qualities of
its own that do something to compensate. The man himself, too—
sportsman, man of the world, excellent comrade, hearty and sincere
good fellow—has been essentially greater in his more recent than in
his earlier times; for the temptations of a success, brilliant and
uninterrupted, did him, as a man at least, little harm. Simple and
generous he was—by all the records of his fellows—when he was at
‘Mr. Sass’s Academy’ fifty years ago. Simple and generous—
generous especially in thought and judgment as well as in action—
he remained, when in the late winter of the present year he was
appointed to the visible headship of the profession to which he had
given so much of the energy of his life.
Sir John Millais was only nine years old when he gained his first
medal at the Society of Arts—Mozart himself scarcely came before
the public in more tender years, as an executant upon the limited
keyboard of his day—and when he was seventeen, ‘Jack’ Millais was
already an exhibitor at the Academy. He was only twenty when his
‘Isabella,’ from the poem of Keats, disclosed a new talent, almost a
new order of talent; at the least, a personality that had to be
reckoned with—an influence that had to be either accepted or fought
against. Yet more marked by an artistic individuality which was, in
part, a return to older conceptions and views than those of his day,
were the ‘Carpenter’s Shop,’ ‘Mariana in the Moated Grange,’ the
‘Huguenot,’ and ‘Ophelia.’ These, or most of them, are typical Pre-
Raphaelite pictures—the offspring of the tacit rebellion of a whole
group of men, only one of whom, Mr. Holman Hunt, remains to give
effect in his later life to the principles enunciated in youth. Dante
Gabriel Rossetti—Pre-Raphaelite to the end, though of course with
certain modifications—was another of those men; but years have
passed since he went from us. The group was completed by others
never as celebrated, nor, as the world judges, so successful. They
painted their pictures; they made their illustrations; they wrote as well
as drew, in the quaint publication called The Germ, which the lapse
of time and the fad of the collector have since made rare and
valuable. Truth, rather than convention, was the aim of their practice;
but they were not peculiar in that,—all youth, if it is earnest at all, is
earnest for truth, or earnest rather for that particular side of truth
which happens just then to have been revealed, and of which it
exaggerates the value. Much has been written about the Pre-
Raphaelite ‘movement’ and its supreme importance—as if it were a
great religious Reformation and a French Revolution rolled into one.
In History it is destined to be remembered because it was a phase
through which two or three men of genius passed—a something,
moreover, that for the moment welded them together. It will not be
recollected, because at a later time mere imitative weaklings, by the
dozen, made feeble fight under what they professed to be its banner.
The interest, then, for sensible people, in Millais’s early pictures,
lies, not in the fact that they were Pre-Raphaelite, but in the fact that
they showed, many of them, an intensity of vision, a profundity of
poetic feeling, which is the property of gifted and of eager youth. The
passionate, constant devotion—the devotion of a minute which lasts,
you feel, for a lifetime; the ‘moment eternal,’ as the great poet puts it
—of the Puritan Maiden and of the Cavalier she helps, is the interest
of the ‘Concealed Royalist.’ The burning love-affair of the ‘Huguenot’
is the interest of a canvas on which, before the days when the
aesthete had invented ‘intensity’ of attitude, Millais had determined
that his lovers should be intense, instead of sentimental. Millais was
in those years occupied very much with the presentation, never of
strictly sensuous enjoyment (Rossetti’s field, rather than his), but of
violent emotion, and uncontrolled, almost uncontrollable, impulse.
His people felt keenly, but with the elevation of poetic natures, or of a
poetic mood. And Millais painted them when their blood ran high. He
chose the incident that seemed to him the most dramatic in all their
story. He painted them on the crest of the wave—at the moment of
crisis.
This, however, like the more naïve Pre-Raphaelitism of a yet
earlier time, was but a phase—remarkable now chiefly because it
has been so absolutely outlived; nay, because so much of the view
of life taken subsequently by its author has, dominating it, a spirit so
opposed to this one. But the transition was not rapid: the ‘Autumn
Leaves’ of 1856, and the ‘Vale of Rest’ of 1860, have, at least, the
poetic quality to the full, though with no violence of emotion. Rather,
they are suggestive and reticent; weird and extraordinarily
expressive: in the one there is depicted the wistfulness of childhood,
in the other the melancholy resignation of a nun to whom ‘rest’
means brooding on a Past more eventful and more poignant than the
occupation of her present day.
Notwithstanding his later technical development, nothing that Sir
John Millais has painted will be remembered more definitely and
firmly than these; and it is noteworthy that they are among the first
pictures in which he relied in great measure upon landscape to
express or suggest the sentiment which it was the picture’s business
to convey. ‘Spring Flowers’ of 1860 was in a lighter and gayer vein, if
it is, as we believe, the picture known originally as ‘Apple Blossom’—
girls lounging in an orchard under the loaded and whitened boughs.
‘My First Sermon,’ in 1863, was more purely popular than anything
we have named. It dealt with childhood almost in the spirit of
Édouard Frère, but with its author’s singular realism of execution.
‘Vanessa,’ in 1869, marked Millais as occupied increasingly with
technical problems—with the attainment of an almost novel boldness
of effect. It is, like so many pieces of his middle and later middle
time, brilliant in colour and brush-work. No one now thinks, we
suppose, of claiming it as dramatic—that is, of connecting it
especially with the character of the lady who came off second-best in
the affections of Swift.
Very soon after the exhibition of ‘Vanessa,’ Millais, who had
already sought impressiveness in landscape background, turned to
pure landscape as a theme sufficient for the exercise of his art. He
gave us then ‘Chill October,’ the October of the north and of the
lowlands, with the wind passing over water, and the reeds and
scanty foliage bent aside by its breath. The picture excited interest. It
was visibly forcible. The conception of the scene, too, was unusual
and, of course, unconventional; but in some later landscape work,
Millais may have been at once nearer to Nature and nearer to the
attainment of a perfected art. ‘New Laid Eggs,’ in 1873, with naïveté
of expression and dexterity of handling, but with a rusticity not very
convincing, was a ‘taking’ picture of happy, healthy, self-confident
girlhood. Its importance, in the volume of its author’s work, was quite
eclipsed the following year by the ‘North-West Passage,’ a canvas
full of interest almost romantic, yet most direct in its record of
character—the main figure being, indeed, a portrait of that Trevelyan
who is associated in most men’s minds with the career of Shelley. He
it was who in Sir John Millais’s picture posed as the sturdy sailor
whose imagination engages him in a remote and unknown voyage.
When, many years after it had been painted, the ‘North-West
Passage’ was seen again in the Millais Exhibition, at the Fine Art
Society’s or at the Grosvenor Gallery, it was felt that at the moment
of its execution the painter had reached the summit of his real artistic
greatness, the masculine and potent hand here best executing that
which had been prompted by a mind at its most vigorous. ‘A Jersey
Lily,’ in 1878, was a tribute to the then girlish beauty of Mrs. Langtry,
who at about the same period was recorded by Mr. Watts with
exquisite simplicity. Again, just as in his diploma picture it had
pleased Millais to invoke the name of Velasquez, and to perform a
feat such as that to which Velasquez was most wont to address
himself, so, in another canvas, in one sense more important—that of
the three Miss Armstrongs playing whist with a dummy—it pleased
him to follow visibly in the steps of Sir Joshua Reynolds—recalling
his composition; the portrait group of the three Ladies Waldegrave
being the one with which he on this occasion made it his business to
vie. In 1879 Sir John was able to exhibit one of the masterpieces of
portraiture—that record or idealisation of Mr. Gladstone of which the
nobility and charm were instantly recognised—a canvas which of
itself would be sufficient to prove that the faculty of poetic vision
never finally deserted an artist who had seemed of late to
concentrate his energy rather on dexterous execution than on the
expression of profound feeling or elevated mood. The ‘Mr. Bright,’
which pretty closely followed the ‘Gladstone,’ was comparatively
unsuccessful. And the illness of the sitter and the consequent
incompleteness of his presentation on Millais’s canvas, made yet
more disappointing the portrait of Lord Beaconsfield which hung
upon the walls of the Academy in 1881. Next year, however, came
the ‘Cardinal Newman,’ to atone for all that had been amiss—again a
poetic vision, a worthy rising to the exigencies of a great theme, a
performance at once decisive and tender, energetic, yet exquisitely
suave.
(Standard, 14th August 1896.)

BURNE-JONES
Unexpectedly and suddenly, from an attack of angina pectoris,
following upon the pest of influenza, Sir Edward Burne-Jones died
yesterday morning. He was sixty-five years old, and he looked worn
for his age—a man of delicate appearance, and certainly of great
sensitiveness; yet, as it had seemed already, of much staying power,
—a ‘creaking gate,’ as his friends thought, not so very regretfully,
since destined, in all probability, to ‘hang long.’ But now his work and
life have been arrested; the laborious days which he had lived for
forty years of manhood are for ever over, and the wan face of the
untiring craftsman, which bent eagerly over his task, and brightened
with quick sensibility in the relaxation of the social hour, is for ever
still. ‘Finis’ is written to the volume of achievement of one of the
greater practitioners in what we may call the second generation of
the English Pre-Raphaelites.
Of the first Pre-Raphaelites—of those of the first generation—
more than one changed his ways, his work, his whole conception of
Art, obviously, as time went on, and the most illustrious of them all—
Millais—was far enough removed from a Pre-Raphaelite in the end.
But of that distinguished and untiring practitioner of the second
generation, whose hold, of late years at least, upon the English and
to some extent upon the French public has become phenomenal,
though it will not be constant, it is certainly to be noted that although
there was, at different times, an unequal capacity, there was at no
time visible change in the direction of his tastes or in the method of
his work. Of the human figure Burne-Jones was not at the first an
excellent, and was never, at any time, an absolutely faultless
draughtsman. Yet the poetry of his figure-drawing, the almost
feminine tenderness with which he followed the lines of dainty
human movement, the dreamy grace that was in the place of
strength, the elegant diffuseness, so to say, which was characteristic
of his style—never even by accident tense and terse—these things
are noticeable in his earlier water-colours and in the very latest of his
performances in this year’s New Gallery. It was as a water-colour
painter that he first began to be known. A pupil of Rossetti, as far as
he was a pupil of any one, Burne-Jones was from the beginning
romantic, and he was affluent in colour.
But what, it may be asked, are the especial characteristics of Sir
Edward Burne-Jones’s art, as it has been revealed not only in the
designs for painted glass, mosaic, tapestry, in numberless pages
decorated with beautiful ornament—such as the Morris translation of
Virgil, and later, the great Chaucer—but likewise in the series of
large pictures, the adequate display of which was, so to say, one of
the raisons d’être of the old Grosvenor Gallery? He had indeed
extraordinary individuality. He was amenable to influence, for all that;
and the influence he felt the most—that of his true fellows—was
exercised by the Italians of the earlier Renaissance: a period
scarcely primitive, scarcely accomplished. Those early Italians,
though engaging, were not really great draughtsmen of the human
figure—not great draughtsmen in the sense of the Greek sculptors,
or Michael Angelo, or Raphael, or Ingres, or Leighton, or
Bouguereau. Sir Edward Burne-Jones, lacking the peculiar
education which fitted the temperament and brought out the qualities
of the men we have named last of all, not unnaturally sympathised
with those in whom intention counted sometimes for more than
execution. But it must not be thought that because the ever-inventive
artist did not possess the Academic qualities, he was not, therefore,
in certain respects, very remarkable in draughtsmanship. He drew
with the ease of conversation; and, though never a master of
accurate gesture—seldom dramatic in the representation of the
particular hour or scene—he was a master of quaint and simple, and
sometimes of elaborate, grace; and for the untiring record of the
particular type of maidenhood, seen best perhaps in the ‘Golden
Staircase,’ or in ‘Venus’s Looking-Glass,’ he stands alone. We name
those pictures rather than, for instance, the ‘Days of Creation,’ or any
of his various ‘Seasons,’ because in them he is at his happiest—his
girls, though in the work of the suave decorator they are never
essentially various, can be radiant as well as doleful. His men have
plenty of wistfulness, but they have rarely energy, strength, decision.
They are even, in a measure, sexless. And of childhood, Burne-
Jones has never been an inspired, or even, it would seem, a
particularly interested chronicler.
Of course, it must be remembered that Burne-Jones is judged
unjustly when judged by the rules of even the least narrow realism.
He painted, not the world of our own day, or of any day—least of all
the Kensington in which he lived, and slept, and had his studio—but
a world he had imagined and created; a world his conception of
which was fed, no doubt, by the earlier and graver of mid-Italian art.
Imagination, now stimulated by legend, now supported by classic
lore, and now the product of the brooding of an isolated mind—that
is really the genesis, the raison d’être, the Alpha and the Omega of
his art. Burne-Jones had, at his best, and especially in his middle
period—the days of the ‘Chant d’Amour,’ with its fitly welcomed
splendours of crimson and blue and golden brown—a wonderful gift
of colour; and, even where the draughtsmanship of the human figure
left something to be wished for, he was a marvellous, a loving, and a
patient draughtsman of flower and of herb. The backgrounds of
some of his inventions, in landscape and the architecture of towns,
were of strange and mystic quaintness. Sometimes, in these, he
recalled almost the spirit, the mystery, almost the charm, of the
backgrounds of the prints by Albert Dürer. The great Dürer!—well,
that is saying much. But we have left to the last what was perhaps
Burne-Jones’s most essential characteristic, certainly his greatest
accomplishment. We mean his gift of composition of line, his power
of precisely and perfectly filling, and never overcrowding, the space it
was his business to occupy. His composition of light and shade was
less remarkable. He was a master of agreeable outline, of flowing
and spontaneous tracery. But if it is not his imagination which is to
keep his memory green, in the minds of the students of Art—and we
doubt whether, with all his very individual merits, it really is—then it is
that in which, in all our generation, and perhaps in all our English
School, he may be accounted to have most possessed—the humbler
faculty of patterning, of weaving faultless webs of subtle line over the
surface, large or small, which was devoted to the exposition of
whatever chanced to be his theme.
(Standard, 18th June 1898.)
BOSBOOM AND HIS
CONTEMPORARIES
The English cognoscenti of the modern type have now for some time
recognised that in Dutch Art there is more than one great period that
has to be reckoned with—that the great Seventeenth Century does
not exhaust the achievements of this people. It may not be quite true
to say of Dutch painting, as of French sculpture, that the traditions
have been invariably preserved, and that there has been little break
in the school; for the last century in Holland was a barren one—just
as barren there as in France and England it was brilliant. The revival
has been for later generations, and of those who did most to
accomplish it some are yet living, in an old age not so very
advanced, and others are lately dead. A history of this revival would
be a great and worthy subject: it may yet, one hopes, be undertaken
by some one writer qualified to treat it. Such a writer could not
possibly be a person who had lived wholly within its influence. He
would have to bring with him something better and wiser than the
ungoverned admirations of the modern studio. A knowledge of the
Past must be his. Meanwhile, we receive, and experience a certain
satisfaction in receiving, even that fragmentary contribution to the
subject which is made in the volume called Dutch Painters of the
Nineteenth Century. Max Rooses—the keeper of the Musée Plantin-
Moretus at Antwerp—furnishes a general introduction, which is
readable and fairly comprehensive, if not particularly critical. And
many writers, whose collaboration is of necessity destructive of unity
of idea, but whose individual opportunities of personal knowledge
give the book something it might yet have lacked had it been written
by one serious and capable critic, contribute biographical notes,
authentic and amiable. The painters have been caressed, not
analysed. That is exactly what the least instructed and least studious
portion of the public is supposed to like, in the ‘text’ of its big
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The ABCs of Gene Cloning

ISBN 978-3-319-77762-7 ISBN 978-3-319-77982-9 (eBook)

Library of Congress Control Number: 2018937521

Printed on acid-free paper

In the 9 years since the First Edition, my contention remains that an

Gene cloning has become a fast growing field with a wide-ranging

Part One. Fundamentals of Genetic Processes

1.1 What Is DNA and What Is a Gene?������������������������������������������ 3

2 Structures of Nucleic Acids 13

4 The Genetic Process 29

6 Reading the Nucleotide Sequence of a Gene 53

Part Two. Techniques and Strategies of Gene Cloning

7 Enzymes Used in Cloning 67

7.3.2 Bacteriophage T4 and T7 Polymerase������������������������ 71

8 Techniques Used in Cloning 75

9 Cloning Vectors for Introducing Genes into Host Cells 93

10.4 The Cloning/Expression Region���������������������������������������������� 125

12 Isolating Genes for Cloning 137

Part Three. Impact of Gene Cloning: Applications in Agriculture

13 Improving Tomato Quality by Antisense RNA 143

14 Transgenic Crops Engineered with Insecticidal Activity 149

15 Transgenic Crops Conferred with Herbicide Resistance 153

16 Growth Enhancement in Transgenic Fish 157

Part Four. Impact of Gene Cloning: Applications

17 Microbial Production of Recombinant Human Insulin 163

18 Finding Disease-Causing Genes 167

19 Human Gene Therapy 177

20 Gene Targeting and Genome Editing 187

20.4 Gene Targeting for Xenotransplants ���������������������������������������� 192

22 Transpharmers: Bioreactors for Pharmaceutical Products 209

24 Whole Genome and Next Generation Sequencing 219

24.4 Strategies For Genome Sequencing������������������������������������������ 224

1.1 What Is DNA and What Is a Gene?

A DNA molecule contains numerous discrete pieces of information,

© Springer International Publishing AG, part of Springer Nature 2018 3

It is therefore more appropriate to define a gene as a functional unit. A

1.2 What Is Gene Cloning?

Gene cloning is the process of introducing a foreign DNA (or gene)

Fig. 1.1. General scheme of gene cloning

1.3 Cell Organizations

Fig. 1.2. Drawing of cells showing details of organelles

Organisms with this type of cellular organization are referred to as pro-

1.4 Heredity Factors and Traits

In a eukaryotic nucleus, DNA exists as complexes with proteins to

Fig. 1.3. Structure of cellular chromosome

In a homologous chromosome pair, the two copies of a gene can exist

The example of round/wrinkled shape of pea seeds is typical of one

1.5 Mitosis and Meiosis

The presence of homologous chromosome pairs is the result of sexual

1.1 What Is DNA and What Is a Gene?�� 3

2 Structures of Nucleic Acids 13

4 The Genetic Process 29

6 Reading the Nucleotide Sequence of a Gene 53

7 Enzymes Used in Cloning 67

7.3.2 Bacteriophage T4 and T7 Polymerase�� 71

8 Techniques Used in Cloning 75

9 Cloning Vectors for Introducing Genes into Host Cells 93

10.4 The Cloning/Expression Region�� 125

12 Isolating Genes for Cloning 137

13 Improving Tomato Quality by Antisense RNA 143

14 Transgenic Crops Engineered with Insecticidal Activity 149

15 Transgenic Crops Conferred with Herbicide Resistance 153

16 Growth Enhancement in Transgenic Fish 157

Part Four. Impact of Gene Cloning: Applications

17 Microbial Production of Recombinant Human Insulin 163

18 Finding Disease-Causing Genes 167

19 Human Gene Therapy 177

20 Gene Targeting and Genome Editing 187

20.4 Gene Targeting for Xenotransplants �� 192

22 Transpharmers: Bioreactors for Pharmaceutical Products 209

24 Whole Genome and Next Generation Sequencing 219

24.4 Strategies For Genome Sequencing�� 224

1.1 What Is DNA and What Is a Gene?

1.2 What Is Gene Cloning?

1.3 Cell Organizations

1.4 Heredity Factors and Traits

1.5 Mitosis and Meiosis

1.6 Relating Genes to Inherited Traits

1.7 Why Gene Cloning?

2.1 5′-P and 3′-OH Ends