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BIOTECHNOLOGY
An Introduction
Second Edition

S. Ignacimuthu, s.j.
 S. Ignacimuthu, s.j.

α
Alpha Science International Ltd.
Oxford, U.K.
Biotechnology: An Introduction
Second Edition
458 pgs. | 65 figs. | 63 tbls.

S. Ignacimuthu, s.j.
Director
Entomology Research Institute
Loyola College (Autonomous)
Chennai, India

Copyright © 2008, 2012

First Edition 2008
Second Edition 2012
ALPHA SCIENCE INTERNATIONAL LTD.
7200 The Quorum, Oxford Business Park North
Garsington Road, Oxford OX4 2JZ, U.K.

www.alphasci.com

All rights reserved. No part of this publication may be reproduced, stored

in a retrieval system, or transmitted in any form or by any means, electronic,
mechanical, photocopying, recording or otherwise, without prior written
permission of the publisher.

ISBN 978-1-84265-754-6

Printed in India
 To
The Society of Jesus
for making me
what I am
 Preface to the
Second Edition

The subject of biotechnology is such a fast growing area that any book written
on this subject has to keep on adding the new developments constantly. The First
edition of ‘Biotechnology: An Introduction’ was well received. Based on the feed-
back received from the readers I have taken efforts to revise the book. Apart from
the addition of all the new developments in biotechnology, three new chapters on
Enzyme and Protein Engineering, Bioinformatics and Nanobiotechnology have
been added. Efforts have been taken to keep the book error-free.
Let me sincerely thank all the readers for their unfailing support and
enthusiasm. Let me also express my gratitude to Narosa Publishing House for
their committed work.

S. Ignacimuthu, s.j.
 Preface to the
First Edition

Biotechnology refers to any technological application that uses biological

systems, living organisms, or derivatives thereof, to make or modify products
or processes for specific use. Biotechnology that is being exploited by many
different sectors. The applications of biotechnology offer enormous potential
for agricultural, pharmaceutical, environmental, healthcare and developmental
purposes.
With the sequencing of the human genome, the researchers are moving to the
next level, which involves understanding the genetic basis of diseases. Benefits
for human development are already experienced. Breakthrough applications in
medicine have huge potential for accelerating human development.
Apart from the usual topics related to biotechnology, aspects related to
Polymerase Chain Reaction, Microarray, Gene targeting, Gene silencing, Animal
cloning, Human cloning, Stem cloning, Genetically modified food, Fermentation
technology, Enzyme technology, Bioinformatics, Drug Discovery, Nano-
medicine, Biosensors and Bioremediation have been elaborated
I am confident that the students and teachers will benefits much from this
book.
S. Ignacimuthu, s.j.
 Acknowledgement

I am grateful to all those who have helped me to prepare this book. My failure
to list their names individually does not detract from my indebtedness to them.
I must thank Sisters Anne Xavier, Sarguna, and Regi, and Miss Parvathy, Holy
Cross College (Autonomous), Tiruchirapalli for correcting the manuscript and
giving the finishing touches. I would also like to specially thank Messrs Albert
Rabara, D. Amalraj, and S. Arockiasamy, St. Joseph’s College (Autonomous),
Tiruchirapalli and Mr C. Muthu, Entomology Research Institute, Loyola College,
Chennai, for their constant and generous help in typing the manuscript. I am
grateful to Rex Johnson for embellishing the book with beautiful illustrations.
I thank my dear students for inspiring me to write this book and also for their
constructive suggestions to improve it. I also thank Dr C.R. Babu, Department of
Botany, University of Delhi, for his guidance and help.
My sincere thanks are due to the following publishers for granting me
permission to use some of their diagrams, explanations, and tables:
Academic Press, New York
Blackwell Scientific Publications, Oxford
Cambridge University Press, Cambridge
Holt, Reinhart and Winston Inc., New York
John Wiley and Sons, New York
Jones and Bartlett Publishers, Massachusetts
Prentice-Hall, International, New Jersey
Springer Verlag, Berlin
W.H. Freeman and Company, New York
Wiley Eastern Limited, New Delhi
Let me thank and compliment the publishers for having published the book in
a neat and excellent manner. Finally, I must acknowledge with deep appreciation
the indispensable help and encouragement received from my dear friends.
 Contents

Preface to the Second Edition vii

Preface to the First Edition ix
Acknowledgement xi

1. Biotechnology – An Overview 1.1

Introduction 1.1
1.1 History 1.2
1.2 Biotechnological processes 1.4
1.3 Products 1.10
1.4 Biotechnology and IPR 1.11
Study Questions 1.14
2. Genetic Engineering and Gene Cloning 2.1
Introduction 2.1
2.1 Outline of a genetic engineering procedure 2.2
2.2 Restriction endonucleases 2.5
2.3 Cloning vehicles or vectors 2.12
2.4 Insertion of a particular DNA molecule into a vector 2.22
2.5 Transformation and growth of cells 2.26
2.6 Detection of recombinant molecules 2.26
2.7 Selection and screening of particular recombinants 2.27
2.8 Genomic DNA libraries 2.28
2.9 Sequencing DNA 2.29
2.10 Gene identification and mapping 2.32
2.11 Analysis of integration and expression of cloned genes 2.34
2.12 Gene amplification and screening 2.36
2.13 Special techniques 2.38
Study Questions 2.42
3. Gene Transfer Mechanisms in Bacteria 3.1
Introduction 3.1
3.1 Transformation 3.2
3.2 Conjugation 3.3
3.3 Transduction 3.8
Study Questions 3.15
xiv Contents

4. Plant Cell and Tissue Culture 4.1

Introduction 4.1
4.1 Historical events 4.2
4.2 Media 4.11
4.3 Plant growth regulators 4.12
4.4 Culture techniques 4.14
4.5 Organogenesis and embryogenesis 4.17
4.6 Special cultures 4.19
4.7 DNA amplification and tissue culture 4.32
4.8 Applications and advances in plant tissue culture 4.35
4.9 Germplasm conservation 4.41
4.10 Trees 4.42
Study Questions 4.46
5. Plant Biotechnology 5.1
Introduction 5.1
5.1 Vectors for plants 5.1
5.2 Ti plasmid based vectors 5.8
5.3 Viral vectors 5.11
5.4 Physical methods of gene transfer 5.16
5.5 In planta transformation 5.20
5.6 Chloroplast transformation 5.21
5.7 Applications of plant biotechnology 5.21
5.8 Uptake of DNA by plant cells 5.27
5.9 Production of disease free and disease resistant plants 5.32
5.10 Viral-resistant plants 5.32
5.11 Insect-resistant plants 5.34
5.12 Herbicide resistant plants 5.35
5.13 Induction and selection of mutants 5.36
5.14 Production through haploid technique 5.41
5.15 Somatic hybrids 5.42
5.16 Transformation through uptake of foreign genome 5.42
5.17 Nitrogen fixation 5.45
5.18 Improving nutritional quality 5.53
5.19 Increased yield of chemical compounds 5.54
5.20 Plants as bioreactors 5.56
5.21 Molecular pharming 5.57
5.22 Antibody production 5.58
5.23 Genetically modified food 5.60
Study Questions 5.64
6. Animal Cell and Tissue Culture 6.1
Introduction 6.1
6.1 Culture 6.2
6.2 Cells in culture 6.3
 Contents xv

6.3 Characterization and validation 6.9

6.4 Cryopreservation 6.11
6.5 Organ culture 6.12
6.6 Animal cell fusion 6.13
6.7 Kinetics of cell growth 6.13
6.8 Culture media for animals 6.15
6.9 Complex natural media 6.19
6.10 Chemically defined media 6.20
6.11 Use of sodium bicarbonate and antibiotics 6.22
6.12 Hybridomas and monoclonal antibodies (MABs) 6.23
6.13 Applications 6.30
6.14 Chimaeric antibodies 6.32
6.15 Hazards associated with MABs 6.33
Study Questions 6.36
7. Animal Biotechnology 7.1
Introduction 7.1
7.1 Vectors for animals 7.1
7.2 Gene-transfer strategies 7.6
7.3 Transient and stable transformation 7.11
7.4 Plasmid vectors for DNA-mediated gene transfer 7.12
7.5 Transgenic animals 7.14
7.6 Applications 7.18
7.7 Human cloning 7.23
7.8 Stem cells 7.24
Study Questions 7.26
8. Industrial Biotechnology 8.1
Introduction 8.1
8.1 Industrial microbial products 8.1
8.2 Industrial plant products 8.16
8.3 Industrial animal products 8.20
8.4 Fermentation or Bioprocess technology 8.30
8.5 Enzyme technology 8.35
Study Questions 8.42
9. Healthcare Biotechnology 9.1
Introduction 9.1
9.1 Production of rare biological molecules 9.1
9.2 Antibiotics, vaccines, and Steroid Hormones 9.3
9.3 Steroid hormones 9.8
9.4 Diagnostic tests 9.9
9.5 Biomarkers 9.15
9.6 Gene replacement therapy 9.18
9.7 Use of nanomedicine 9.25
 xvi Contents

9.8 Stem cell therapy 9.27

9.9 Monoclonal antibody therapy 9.27
Study Questions 9.31
10. Environmental Biotechnology 10.1
Introduction 10.1
10.1 Waste treatment 10.1
10.2 Biomass production 10.10
10.3 Biodiesel production 10.17
10.4 Biodiversity 10.18
Study Questions 10.22
11. Bioinformatics 11.1
Introduction 11.1
11.1 Important contributions 11.2
11.2 Sequencing development 11.5
11.3 Aims and tasks of bioinformatics 11.7
11.4 Application of bioinformatics 11.7
11.5 Challenges and opportunities 11.10
11.6 Drug discovery 11.11
11.7 Pharmainformatics 11.15
11.8 Search programs 11.16
Study Questions 11.20
12. Nanobiotechnology 12.1
Introduction 12.1
12.1 Nanomaterials and nanoparticles 12.1
12.2 Biomaterials 12.2
12.3 Drug delivery 12.3
12.4 Tissue engineering 12.6
12.5 Nanomedicine 12.8
12.6 Biosensors 12.11
12.7 Nanobiosensors 12.15
12.8 DNA Nanotechnology 12.18
Study Question 12.22
13. Enzyme and Protein Engineering 13.1
Introduction 13.1
13.1 Principles of enzyme and protein engineering 13.2
13.2 Assumptions for enzyme and protein engineering 13.2
13.3 Methods for enzyme and protein engineering 13.3
13.4 Enzyme and protein technology 13.4
13.5 Artificial enzymes 13.8
13.6 Genetic engineering of enzymes and proteins 13.11
13.7 Applications of enzymes 13.13
Study Questions 13.18
 Contents xvii

14. Biotechnology and Ethics 14.1

Introduction 14.1
14.1 Medical and chemical biotechnology 14.2
14.2 Agriculture and food 14.2
14.3 Energy and environment 14.3
14.4 Humans 14.5
14.5 Bioethics: facing problems and finding solutions 14.11
Study Questions 14.14
Further Reading FR.1
Glossary   G.1
Index I.1
 1 Biotechnology—
An Overview

Introduction
Biotechnology has been defined in various ways. In simple terms, biotechnology
refers to the use of living organisms or their products for the welfare of humanity.
One rather vague definition says that biotechnology is the application of biological
organisms, systems or processes to manufacturing and service industries. In other
words it simply means biology applied for use.
According to the definition adopted by the European Federation of
Biotechnology, created in 1978, ‘biotechnology makes it possible, through an
integrated application of knowledge and techniques of biochemistry, microbiology,
genetics and chemical engineering, to draw benefit, at the technological level, from
the properties and capacities of microorganisms and cell cultures. Biotechnology
offers the possibility of producing, from widely available renewable resources,
substances and compounds essential to life and the greater well-being of human
beings.’ In short, biotechnology comprises technical processes that enable us not
only to manipulate the DNA, but also to use in other ways living organisms for
specific purposes. Biotechnology is defined as the application of current scientific
methods and techniques to improve the biological systems, be they plants, animals,
microorganisms, for the betterment of human beings.
The term biotechnology was brought into popular use in the mid-1970s as
a result of the increased potential for the application of the emerging techniques
of molecular biology. The word itself seems to have been first employed by the
Leeds City Council in the United Kingdom in the early 1920s, when they set
up an Institute of Biotechnology. In fact, biotechnological processes are nearly
5000 years old. It began with the discovery of fermentation and the consequent
production of alcoholic beverages. Present interest in biotechnology has been
stimulated by the potential that can result from the marriage of biological
processes and techniques—some old, some new—with production engineering,
electronics and bioprocessing.
 1.2 Biotechnology

1.1 History
Probably the oldest biotechnological processes are found in microbial
fermentations, as borne out by a Babylonian tablet dated around 6000 B.C.,
unearthed in 1981, depicting the preparation of beer. The Sumerians were
able to brew as many as 20 different varieties of beer in the third millennium
B.C. The perfecting of fermentation processes, their increased efficiency,
and the discovery of a large number of microbial bioconversions, along with
the isolation of substances of bacterial and fungal origin to replace synthetic
products has led to the discovery of more effective drugs and medicines. Recent
biotechnological processes rely on genetic recombination techniques as well as
the use of immobilized enzymes, cells or cell organelles.
At this stage, a brief history of various events that led to the present-
day knowledge of biotechnology will be useful. The foundation of modern
biotechnological applications can be traced to 1866 when Czech monk Gregor
Mendel published the results of his experiments on garden pea. He suggested
the involvement of some factors in the transfer of traits from one generation
to another. Later this factor was determined as the gene. After the rediscovery
of Mendelism in 1900, biologists were concerned mainly with the inheritance
of structural or other visible variations. Later, A.E. Garrod (1902) recognized a
class of defects in human beings such as diabetes, phenylketonuria, tyrosinosis
cretinism, albinism etc., which were caused by a fault in the metabolism within
the body.
O.T. Avery, C.M. MacLeod and M. McCarty (1940) were the pioneers in
studying the chemical nature of the substance that was responsible for bacterial
transformation. G.W. Beadle and E.C. Tatum (1941) carried out genetic experiments
with the bread mould Neurospora crassa with a biochemical slant, and established
that genes worked through biochemical pathways. They also postulated that each
gene was responsible for the synthesis of one particular enzyme.
The whole structure of a protein, insulin, was established by Sanger (1953).
Crick and Watson (1953) showed that deoxyribonucleic acid (DNA) had a
double-stranded structure. Nirenberg (1963) deciphered the genetic code that was
applicable from bacteria to man. Merrifield (1963) manufactured and marketed
the first automatic polypeptide synthesizing machines. Edman and Begg (1967)
developed methods for protein degradation. Arber, Smith and Nathans (1972)
discovered the restriction enzymes which cut out DNA at specific points. Gilbert,
Maxam and Sanger (1976) developed rapid methods for chemical analysis of
DNA. Itakura and his co-workers (1977) synthesized the genes of human
somatostatin and insulin. N. Goodon and M.D. Chilton (1977) proved that the
transfer of genes was possible using the bacterium Agrobacterium tumefaciens
as a carrier H.G. Khorana (1979) succeeded in synthesizing, for the first time, an
entirely artificial gene capable of functioning within a living cell. Itakura (1980)
also constructed the first gene assembler. Hood (1981), who invented the protein
micro-analyser, built another automated machine for the same purpose.
 Biotechnology—An Overview 1.3

Kary Mullis (1983) invented Polymerase Chain Reaction (PCR) which

revolutionized biotechnological applications. Alec Jeffrey (1984) developed
genetic fingerprinting technique which can be used to identify individuals
by analysing the varying sequences (polymorphisms) in the DNA. The
human genome project was initiated in 1986. In 1995 M. Sehena developed
complementary DNA microarray system to monitor gene expression; also the
Institute for Genomic Research reported the first complete DNA sequence of the
genome of a free-living organism. In 1997 Dolly the sheep was cloned using
somatic cell by Ian Wilmut and his colleagues. Also the complete sequence of
the genome of the yeast Saccharomyces cerevisiae was reported. In 1999 human
chromosome 22 was sequenced. In 2000, the genome sequence of Adabidopsis
thaliana was completed. In 2001, complete map of the genome of rice was
reported. Also annotations and analysis of human genome was published. In
2002, cloned pigs were reported. In 2003 human stem cells were used to treat
diseases. Mouse genome was also sequenced.
In the field of tissue culture, isolation of protoplast was carried out by
Klercker (1892). Haberlandt (1902) demonstrated the totipotency of cells. White
(1934) showed the possibility of growing excised tomato root tips in vitro for
an indefinite period. Gauthert (1937) succeeded in cultivating undifferentiated
carrot tissues. Van Overbeek and his group (1941) isolated embryos of Datura
which could grow and develop on a chemical medium supplemented with
coconut milk. The possibility of regenerating plants in vitro from the shoot apex
of certain angiosperms was first demonstrated by Ball (1946). Skoog and Tsui
(1948) showed that shoot initiation, also termed as caulogenesis, in tobacco
stem segments and callus could be chemically regulated by manipulating the
nutrient medium. La Rue (1949) managed to grow immature maize endosperm
in culture.
Morel and Wetmore (1951) were the first to achieve success with monocot
cultures. Tulecke (1953) obtained the first haploid callus from the pollen grains
of Ginkgo biloba. Munir, Hildebrandt and Riker, (1954) reported the growth
of isolated cell cultures in a liquid medium. Skoog’s group (1955) identified
6-furfurylaminopurine, a degraded product of herring sperm DNA, as a chemical
capable of stimulating cell divisions. Skoog and Miller (1957) showed that shoot
and root initiation in tobacco callus cultures could be regulated by maintaining
a subtle ratio between auxin and cytokinin in the medium, paving the way for
the chemical regulation of organogenesis. Carew and Schwarting (1958) were
the first to get callus cultures from rye, a monocot. Steward (1958) discovered
the differentiation of somatic bipolar embryos in carrot using cell suspension
techniques. Reinert (1959) reported the use of a nutrient medium solidified with
agar for embryogenesis.
Cocking (1960) isolated plant protoplasts enzymatically. Morel (1960)
developed the techniques of shoot apex culture of orchids for clonal propagation.
Guha and Maheshwari (1964) obtained direct embryos from cultured anthers of
 1.4 Biotechnology

Datura which led to the development of haploid plants. Nishi and his colleagues
(1968) were the first to induce differentiation in monocot callus culture of rice.
Binding and his colleagues (1970) isolated streptomycin resistant callus of
Petunia hybrida. Carlson and his group (1972) were the first to fuse the protoplast
of Nicotiana glauca and Nicotiana langsdoiffti, two sexually compatible species
of tobacco, and regenerated a parasexual hybrid.

1.2 Biotechnological Processes

The development of biotechnology relies to a large extent on the existence of
effective research in microbiology, biochemistry, enzymology and microbial
genetics as well as on the existence of culture collections, perfectly recorded and
regularly studied. The use of animal cells also plays an important role, such as
in the culture of viruses for producing vaccines, in the production of interferons
and in the synthesis of monoclonal antibodies by hybridomas. Plants also have
contributed in the production of plant clones and synthesis of various alkaloids
and other secondary metabolites. Microbiology, genetics, molecular biology and
biochemistry are the roots of biotechnology.
Activity in biotechnology may be conveniently broken down into eight
areas of endeavour, namely, (i) Recombinant DNA and genetic engineering, (ii)
cell cultures (iii) waste treatment and utilization, (iv) enzymes and biocatalysts,
(v) fuels, (vi) nitrogen fixation (vii) fermentation and pharmaceuticals and (viii)
healthcare (Table 1.1).

Table 1.1 Areas in biotechnology and the principal products

Technology Products
(i) Recombinant DNA and Enzymes, Vaccines, Interferons, hormones,
Genetic Engineering antibodies, blood factors.
(ii) Cell cultures Biomass production, single cell proteins, fine
chemicals, interferons, vaccines, blood products,
monoclonal antibodies.
(iii) Waste treatment and Byproduct utilization, e.g. cheese whey, waste
utilization cellulose, recovery of catalyst, water recycling.
(iv) Enzymes and Food processing, fine chemicals, diagnostic kits,
biocatalysts chemotherapy, biosensors, isoglucose, glucose
syrups, ethanol.
(v) Fuels Alcohol, hydrogen, methane, gasohol
(vi) Nitrogen Fixation Reduction of nitrogenous fertilizers
(vii) Fermentation and Alcohols, fine chemicals, antibiotics, vitamins,
Pharmaceuticals enzymes, amino acids, nucleotides, steroids,
alkaloids, diagnostic reagents, citric acid,
biopolymers, biopesticides, ethanol, acetone,
butanol, biogas.
(viii) Healthcare Antibiotics, hormones, DNA probes, gene therapy.
 Biotechnology—An Overview 1.5

1.2.1 Recombinant DNA and Genetic Engineering

Since 1972 technology has, however, been available that allows the identification
of genes for specific, desirable traits and the transfer of these, often using a virus
as the vector, into another organism. Comparable to a word-processor’s ‘cut-
and-paste’, this process is called recombinant DNA technology or gene splicing.
Virtually any desirable trait found in nature can, in principle, be transferred
into any chosen organism. An organism modified by gene splicing is called
transgenic or genetically modified (GM). Specific applications of this type of
genetic engineering are rapidly increasing in number - in the production of
pharmaceuticals, gene therapy, development of transgenic plants and animals,
and in several other fields.
The ability to isolate a gene coding for a desired product and transfer it to
another organism has opened the way either to the more effective production
of useful proteins, or to the introduction of novel characteristics in the host
organism. Thus the large-scale production of hormones, vaccines, blood clotting
factors or enzymes by some friendly bacterium has become possible.
There is also an additional possibility with this form of technology, i.e. the
production of truly novel proteins. By selectively modifying the gene coding for
an enzyme, before it is introduced into the host organisms, its structure, and hence
the properties of the enzyme may be advantageously modified. Modification of
the genome of economically important plants is also very promising. The levels
of storage proteins in seeds could be increased to give high protein seeds. It is
also possible that crops could be engineered for a greater resistance to herbicides
or infection.

1.2.2 Cell Cultures

The problem of culturing mammalian cells on a large scale will become a major
preoccupation of cell biologists and biochemical engineers in the near future.
Monoclonal antibodies, due to the complexity of the transcription and translation
of their genetic material, and interferons, due to the cost and effectiveness, are
likely to become more important in future both for therapeutical, preparative and
analytical applications.
Plants are an important source of many valuable raw materials and high-
value drugs. The ability to culture plant cells on a large scale, either for the
production of biomass per se or in order to extract the desired product from cell
cultures, is becoming a highly desirable technology. Immobilization technology
has come in handy to synthesize complex compounds and to produce greater
amounts of secondary metabolites.

1.2.3 Waste Treatment and Utilization

Sewage disposal is a problem long faced by human beings. There are many
forms of wastes which cannot only be more easily disposed of, but can perhaps
 1.6 Biotechnology

be turned into a useful commodity. For example, cheese production starts with
the curdling of milk to form the solid curds and liquid whey. An average size
cheese making plant produces thousands of litres of whey a day, and letting it go
waste in the sewers is not only problematic but also costly. Whey is composed
of a few proteins, minerals and about 4% lactose. If these could be broken down
to useful compounds with the help of engineered enzymes, then it would help in
waste disposal.
Cellulose is another abundant waste material, particularly in the form of
straw from cereal crops. Traditionally, it is either ploughed back into the fields
or burnt. In theory, this waste cellulose could be biologically degraded and used
as a feedstock for the production of microbial proteins. It has been estimated that
sufficient protein could be produced in this way from agricultural wastes alone
to feed the entire world’s population. Also other waste materials like herbicides,
pesticides, chemicals etc. used in agriculture could be degraded into useful by-
products.

1.2.4 Enzymes and Biocatalysts

Enzymes are nature’s supreme catalysts, exhibiting great specificity and
enormous catalytic power. They have been in use for many centuries, particularly
in food processing (for example cheese making, removal of hair from hide etc.)
and represent one of the oldest forms of biotechnology. More recently, great
interest has been shown in extending the use of enzymes (whether purified, as
dead or partially viable cells) in food processing, chemical production, analytical
and diagnostic systems, and in the treatment of diseases. The elucidation of the
structure and functions of certain enzymes as well as their use, in an immobilized
form, in a variety of industrial production processes has opened great avenues for
human endeavour and curiosity.

1.2.5 Fuels
The world is running short of combustible fuels, particularly, mineral oils.
Biotechnology might offer new fuels and alternative carbon feedstocks. Soon
we may use methane, biodiesel and hydrogen as important fuels. Biophotolytic
production of hydrogen from water has already been achieved, and is based on
the combination of the photosystems of plants with bacteria-derived hydrogenase
enzymes and light. This seems to be the ideal combustible fuel since it produces
no pollution and regenerates its source material.

1.2.6 Nitrogen Fixation

The introduction into crops the ability to fix nitrogen from the atmosphere
would not only save on the cost of applying nitrogenous fertilizers, but would
eliminate the potential problems of water pollution from nitrates washed off from
agricultural land. The achievement of such associations between nitrogen-fixing
 Biotechnology—An Overview 1.7

bacteria and cereals would have important agricultural applications. Attempts

are being made to increase the efficiency of nitrogen fixation and assimilation by
acting on the genetic mechanisms regulating the process. Research is going on to
develop new nitrogen fixing systems by means of somatic hybrids between the
desired cultivars and nitrogen-fixing plants; or by introducing the nif genes into
symbiotic or free living microorganisms; also to transfer the nitrogen-fixing ability
to plants by using viruses or self-transmissible plasmids as vectors of the nif genes;
or by recombining these genes with mitochondrial or chloroplast DNA.

1.2.7 Fermentation and Pharmaceuticals

Fermentation along with biocatalysts shares the distinction of being the oldest form
of biotechnology. Traditionally, fermentation has meant the production of potable
alcohol from carbohydrates. However, fermentation, that is, the application of
microbial metabolism to transform simple raw materials into valuable products
can produce an amazing array of useful substances; for example, chemicals such
as citric acid, antibiotics, biopolymers, single cell proteins, vitamins, alkaloids,
steroids, vaccines and other diagnostic reagents can be produced using the
fermentation technology.
It is one thing for the laboratory scientist to clone a novel gene, discover
a new antibiotic or invent an enzyme catalysed process, but it is quite another
to transfer this knowledge to the scale of operation required to make a useful
product in significant amount. Process engineers play a vital role in this transfer
by employing various techniques such as harvesting, pretreatment, filtration
of the raw material, reactor design, reuse of biocatalyst or organisms, product
extraction and analysis. And this makes biotechnology worth billions of dollars
by contributing to the gross domestic product of the economy in many developed
countries. In terms of national economic growth as a result of the introduction of
biotechnology, the greatest potential lies in the food and chemical industries.

1.2.8 Vaccines: Recombinant Technology and the Immune

System
A vaccine is an antigen, e.g., the surface proteins of a pathogenic microorganism.
Recombinant technology constitutes a powerful tool for the production of purer
and safer vaccines. For example, the insertion of a hepatitis B virus gene into the
genome of a yeast cell allows the production of pure hepatitis B surface antigen
- a very effective vaccine, biologically equivalent of an inactivated vaccine.
A live attenuated typhoid vaccine is now being produced from a Salmonella
typhi bacterium cell line modified by recombinant technology so as not to
cause typhoid. Several new vaccines using genetically weakened versions of
microorganisms for which vaccines have either not existed before or been only
marginally effective, are now making their way through the testing process.
Separately, recombinant technology is now being used to modify plants,
rather than animal cell lines or microorganisms, to produce vaccines. Likely to
 1.8 Biotechnology

gain increased use in the future, this will enable many vaccines to be made for
oral administration, thus overcoming many vaccine logistics constraints and the
need for medically qualified or veterinary personnel and other costly elements
currently necessary to carry out effective immunisations. The first potato-
produced, edible hepatitis B vaccine is in clinical trial.
In addition to vaccines to prevent against microorganisms, others – so-
called therapeutic vaccines - based on combining immune pathology and genetic
modification may soon revolutionise the treatment of many diseases – infectious as
well as non-infectious. Some of these will stimulate an impaired immune response
in an individual who is already infected with that organism and has mounted
an inadequate immune response to that organism. The aim of administering a
therapeutic vaccine may be to increase the individual’s immunity to an organism
that, for instance, is unable to provoke an appropriate response on its own. Similarly,
vaccines are being developed for use in the treatment of diseases.

1.2.9 Monoclonal Antibodies

While, vaccines are antigens which, when inoculated, cause the immune system
to produce antibodies, recombinant technology is being used, as well, to produce
antibodies directly. In this variation on the immune/genetics theme, single cell
lines, i.e. cloned, wholly identical, specialised cells that can be grown indefinitely
are used to produce antibodies of singular specificity - monoclonal antibodies.
These are used in a number of diagnostic applications, as well as to prevent acute
transplant rejection, and treat leukaemias and lymphomas. Some show promise
against auto-immune diseases.

1.2.10 Gene Therapies

While the above applications mostly rely on using modified organisms or cell
lines to produce substances in vitro that can then be used to treat or prevent human
disease, gene therapy is distinctly different in that it essentially modifies the
patient’s own genetic setup. In other words, while the aim remains the manipulation
of a specific gene into a designated host cell, the ‘host’ is a ‘population’ of cells
in situ in the human body. In contrast to the above technologies, gene therapy
takes place in vivo.
Gene therapy essentially makes use of an approach similar to recombinant
technology. An isolated gene encoding for the desired characteristic is spliced
into the genome of a virus, often itself modified so as not to cause disease.
Infecting the host organism, the virus introduces the gene into the target cells to
‘appropriate’ the cells protein-making apparatus.
Gene therapy was first used in 1990, for an enzyme deficiency. Since then,
more than 100 clinical gene-therapy trials have been initiated world-wide. Most
of the trials have been for the treatment of tumours (predominantly malignant
melanoma and haematological disorders), but there have also been trials of gene
 Biotechnology—An Overview 1.9

therapies for genetic disorders, AIDS, and cardiovascular disease. While many
technical problems are yet unsolved, in relation to vector design as well as to
clinical safety and efficacy, gene therapy appears likely to become an important
part of the armoury with which disease will be fought in the future.

1.2.11 Stem Cells

Upon fertilisation, an egg cell initially starts dividing into undifferentiated cells
from which, later, cells of increasing specialisation develop and from which
eventually the highly differentiated cells in tissues of different organs stem. In
human embryos, the potential for giving rise to cells of any specialisation is held
only by very early, primitive, so-called totipotent stem cells, at the most up to the
16 cell stage. Identical twins (triplets etc.) originate from totipotent cells, i.e. the
result of a cleavage of the embryo within a few days after fertilisation.
At the next stage of development, the now pluripotent stem cells have
already acquired some degree of specialisation. While they are no longer
individually able to give rise to a foetus, they are still able to differentiate into
any cells of an adult human being. Multipotent stem cells can be derived from
foetuses or umbilical cord blood, and are even present throughout life, although
in progressively decreasing numbers in adults. Unless ‘reprogrammed’, the
latter cells are probably only able to develop into specialised tissues or organs.
Common to stem cells is their ability - under given circumstances - to multiply
almost indefinitely and be stimulated to grow into a variety of specialised tissues,
opening up vast possibilities of tissue repair.
The potential scope of stem cell research and derived applications is
enormous such as renewing heart muscle in congestive heart failure; replacing
blood-forming stem cells to produce healthy red and white blood cells to treat
e.g. AIDS and leukaemias; relining blood vessels with new cells as treatment
for atherosclerosis, angina, or stroke; restoring islet cells in the pancreas to
produce natural insulin in diabetics; or renewing of nerve cells in patients with
Parkinson’s disease or paralysis.

1.2.12 Cloning
A clone is essentially the result of asexual reproduction, leaving clones with
no choice but to accept a genome identical to that of their ancestor. Microbes
reproduce by cloning; the chrysanthemum plants are clones of a long dead plant.
And one of a pair of identical twins is a clone of the other. Cloning in modern
biotechnology is based on cell nucleus transfer Cloning a mouse, a mammal far
better known as a laboratory animal than sheep, was tried unsuccesfully for a
long time and, Dolly was the only success among about 300 attempts. Most of the
attraction for cloning derives from its potential in pharmaceutical production. Of
particular allure is the potential of having animals express proteins of therapeutic
value in the milk.
 1.10 Biotechnology

1.2.13 Pharmaceutical Production

The first major healthcare application of recombinant technology was in the
production of human insulin, a hormone substantially involved in the regulation
of metabolism, particularly of carbohydrates and fats, and the relative lack of
which leads to the clinical condition called diabetes mellitus. Insulin is a relatively
small protein consisting of 51 amino acids.
A perhaps more famous example is recombinant erythropoietin, a hormone
that regulates the production of red blood cells. The clinical conditions for which
erythropoietin is indicated are relatively rare, but the bio-engineered product has
gained enormous popularity in professional sports – as EPO – because it enables
athletes to add 15-20 per cent to their oxygen carrying capacity.
Using microorganisms or human cell cultures, similarly modified, in the
production of highly complex molecules which would otherwise be impossible,
or extremely difficult, to synthesise, is now employed extensively by the
pharmaceutical industry. Increasingly, higher animals - “bioreactors” – modified
by recombinant technology and able to express high value pharmaceutical
proteins in their milk are also gaining use in reducing the cost of creating and
producing new medical products.

1.2.14 Healthcare
Biotechnological inputs have been helpful in i) preventing diseases through
various therapeutic products like antibiotics, antibodies, vaccines, hormones,
regulatory products, etc., ii) prenatal diagnosis of genetic diseases and genetic
counseling, iii) immunodiagnostic tools and probes aiding disease identification,
iv) correction of diseases, v) personalized medicine, vi) gene therapy, vii)
cloning, etc.

1.3 Products
Biotechnology has promoted the production of monoclonal antibodies, DNA
probes, antibodies, antibiotics, recombinant vaccines, human insulin, interferons,
human growth hormones, which are helpful in the treatment of various diseases
and metabolic disorders. On the industrial front, production of ethanol, lactic
acid, citric acid, glycerine, acetone, penicillin, streptomycin, erythromycin,
cycloheximide, immunotoxins, amylase, protease, lipase, single cell proteins has
been taken up in a large-scale. On the agricultural front, production of many
transgenic plants and clonal multiplication has been done. On the environmental
front production of biosensors and bioplastics has been going on.
In the year 2000, the global revenues of biotechnology industry were estimated
to be more than US$ 70 billion, with biopharmaceuticals forming the largest
chunk accounting for 60% of the market followed by diagnostics (14%) and
industrial enzymes (4%). Therapeutic proteins accounted for 10% of the global
 Biotechnology—An Overview 1.11

pharmaceutical market. In India, the revenue of biotech industries for 2004-2005

is as follows: Biopharma- 35700 million; Bioservices- 4250 million; Bioagri-
3300 million; bioindustrial-3200 million and bioinformatics- 800 million.
The development of biotechnology will be a long-term affair, dependent on
the needs of market forces, the acceptability of the products and developments in
competing technologies. The key to the successful development of biotechnology
lies either in producing a product which can be made by no other means, or by
producing an existing product more cheaply and abundantly. The latter approach
involves catching up on existing technology, which itself may be advancing,
and thus involves considerable risk. Some possible medium-term products such
as vaccines for common cold, safer tobacco substitutes, cheap wines, reliable
self-diagnosis kits, site specific drugs etc. which are possible only through
biotechnology may eventually fulfill many dreams.
The spectacular development of biotechnology is full of promises. In addition
to the contribution at the family, village and industrial levels biotechnology offers
some solutions to humanity as a whole by tackling the problems of food and
protein deficiencies. It may also contribute to the therapeutic revolution that will
rely on the progress of cellular biochemistry, molecular biology and immunology,
and will call more upon biological substances to fight against diverse pathological
problems. Biotechnology also contributes to the preservation of genetic diversity
as well as to technological innovations.

1.4 Biotechnology and IPR

Intellectual Property Rights (IPRs) are essential part of today’s business. IPR’s
are the means to protect any intangible asset. Examples of IPR are patent,
copyright, trademark, geographical indication and trade secret. A patent is an
exclusive monopoly granted by the government to an inventor over his invention
for limited period of time. Some examples are given below:
Bayer Crop Science GmbH (Frankfurt, Germany) has patented nucleic acid
molecules which encode enzymes involved in starch synthesis in plants. These
enzymes are wheat isoamylases. The invention furthermore relates to vectors and
host cells which contain the above-described nucleic acid molecules, in particular
to transformed plant cells and plants which can be regenerated from these and
which have an increased or reduced activity of the isoamylases according to the
invention. (US 6,951,969)
Columbia University (New York, NY) has patented antibodies directed to
OLD-35 protein, the product of the OLD-35 gene, which displays enhanced
expression during cellular senescence and terminal cell differentiation. (US
6,951,923)
Ruan, et al. has patented a “cocktail” combination of two monoclonal
antibodies respectively acting on different sites of the platelet GPIIb-IIIa
complex. This “cocktail” combination can completely block receptor function
 1.12 Biotechnology

of the GPIIb-IIIa complex, inhibit platelet aggregation and thereby efficiently

inhibit thrombosis. (US 6,951,645)
University of California (Oakland, CA) and Chiron Corporation (Emeryville,
CA) has patented gene delivery vectors, such as, for example, recombinant
adeno-associated viral vectors, and methods of using such vectors for use in
treating or preventing diseases of the eye. (US 6,943,153)
Deltagen, Inc. (San Carlos, CA) has patented nucleotide constructs and
methods for making DNA constructs useful for introducing sequences into and
disrupting the function of a gene in a cell, particularly an embryonic stem cell.
(US 6,942,995)

Study Outline
Biotechnological Processes
Biotechnology is the application of biological organisms, systems or processes
to manufacturing and service industries. The major areas of interest in this field
are genetic engineering, Recombinant DNA, culture techniques, waste treatment,
utilization of enzymes and biocatalysts, fuel, nitrogen fixation, fermentation
and pharmaceuticals and healthcare. The extraction and transfer of a gene with
the desired characters, has been a fascinating field of achievement and this has
resulted in the production of useful proteins and novel characters. Large scale
production of hormones, vaccines and bacteria are noteworthy achievements.
Microbial fermentation is considered to be one of the oldest biotechnological
processes and has captured the attention of modern biotechnologists who had
given this a highly industrial status due to the various products that are formed
as a result of it.

Cell Culture
The ability to culture plant cells on a large scale and protoplast isolation and
fusion have given a boost to agriculture. Newer varieties which are early fruiting
and which supply better fruits, owe their credit to biotechnology. Immobilization
technology has come in handy to synthesize complex compounds and to
produce greater amount of secondary metabolites. Technological selection and
modification of genome and its introduction into crops have helped to synthesize
novel proteins and increased the nitrogen fixing ability of plants.

Waste Treatment and Utilization

Sewage disposal is a problem long faced by mankind. Today there are many forms
of waste which are turned into useful products with the help of biotechnology.
Whey, which results from cheese industry is considered to be a waste product. It is
composed of proteins, minerals and about 4% lactose. With the help of engineered
enzymes these could be broken down to useful compounds. Similarly cellulose is
biodegraded and is used as a feedstock for the production of microbial proteins.
 Biotechnology—An Overview 1.13

Enzymes are nature’s supreme catalysts and exhibit great specificity. Recently
great interest has been aroused in using these in food processing, chemical
production, analytical and diagnostic systems and in the treatment of diseases.
Biotechnology has offered new fuels and alternative carbon feedstocks.

Nitrogen Fixation
Introduction of nitrogen fixing ability into nonleguminous crops through transfer
of nif genes is another field and if it achieves total success, it will be a great boon
to agriculture. Research is going on to develop new nitrogen fixing systems by
means of somatic hybrids between the desired cultivars and nitrogen fixing plants;
or by introducing the nif genes into symbiotic or tree living microorganisms.

Fermentation, Pharmaceuticals and Healthcare

Application of microbial metabolism to transform simple raw materials into
valuable products can produce an amazing range of useful substances; e.g.,
chemicals such as citric acid, antibiotics, biopolymers, single cell proteins,
vitamins, alkaloids, steroids and other diagnostic reagents. The development of
biotechnology, therefore, relies to a large extent, on the existence of effective
research in microbiology, biochemistry, enzymology, microbial genetics, cell
cultures and molecular biology. Biotechnological inputs have been helpful in
preventing diseases and in identifying and treating many diseases.

Vaccines
Vaccines are antigens. Recombinant technology is a powerful tool for the
production of purer vaccines. Several new vaccines are produced using this
technology. Plants are also used for the production of edible vaccines. Vaccines
will also stimulate an impaired immune response.

Monoclonal Antibodies
Clones, wholly identical specialized cells that can be grown indefinitely are
used to produce antibodies of singular specificity. They are used in a number of
diagnostic applications.

Gene Therapies
Gene therapy modifies the patient’s own genetic setup. An isolated gene is spliced
into the genome of a virus and allowed to infect the host organisms to introduce
the desired gene and correct the defect.

Stem Cells
Stem cells are unspecialized and undifferentiated cells with potential to give rise
to many types of specialized cells. Embryonic stem cells and adult stem cells are
available. They have many useful applications.
 1.14 Biotechnology

Cloning
A clone is an identical copy of the other. A clone results from asexual reproduction.
Cloning now is based on cell nuclear transfer. Cloning is helpful in producing
animals for pharmaceutical purposes.

Biotechnology and IPR

Intellectual Property Rights (IPR) are essential part of today’s business. They
are a means to protect any intangible asset. Examples are patent, copyright,
trademark, geographical indication and trade secret.

Questions
1. Define biotechnology?
2. What is recombinant DNA technology?
3. What are the roles played by enzymes and biocatalysts in biotechnological
process?
4. What role does biotechnology play in waste treatment and utilization?
5. What are the various fermentation products?
6. Describe vaccine, monoclonal antibody, gene therapy, stem cell and
cloning.
7. What is IPR?
 2 Genetic
Engineering and
Gene Cloning

Introduction
Genetic engineering involves a manipulation of the genetic material towards
a desired end in a directed and pre-determined way. This is alternately called
recombinant DNA technology or gene cloning. Strictly to ‘engineer’ means to
design, construct and manipulate according to a set plan. Genetic engineering
aims at isolating DNA fragments and recombining them. The basic technique
is quite simple. Two DNA molecules are isolated and cut into fragments by
one or more specialized enzymes and then the fragments are joined together in
a desired combination and restored to a cell for replication and reproduction.
The term recombinant DNA is also used specifically to refer to composite DNA
molecules, that result from the physical combination of DNA segments derived
from different sources.
Genetic engineering started developing in the mid-1970s when it became
possible to cut DNA and to transfer particular pieces of DNA containing specific
bits of information, from one type of organism into a second type of organism. As
a result, the characteristics of the second organism (recipient) could be changed
in a specific and pre-determined way. When the recipient organism is a microbe,
such as a single celled bacterium, the specific fragment of transferred DNA is also
multiplied many times as the recipient microbe multiplies. Millions of identical
cells, i.e. a clone of cells, eventually arise. Consequently, it is possible to obtain
millions of copies of a specific region of DNA inside a bacterial cell by allowing
the cell (and the piece of DNA) to multiply millions of times.
Current interest in genetic engineering is due to its varied applications such
as: 1. isolation of a particular gene, part of a gene, or region of a genome, 2.
production of a particular RNA and protein molecules in quantities formerly
thought to be unobtainable, 3. improvement in the production of biochemicals
(such as enzymes and drugs) and commercially important organic chemicals,
4. production of varieties of plants having particular desirable characteristics
 2.2 Biotechnology

(e.g., requiring less fertilizer or resistant to disease etc.), 5. correction of genetic

defects in higher organisms, and 6. creation of organisms with economically
important features (e.g., plants capable of maturing faster or having greater
yield). All these are made possible by some basic methodologies which form the
essential ingredients of genetic engineering. They are:
(i) a method for physically joining two DNA segments together,
(ii) a self-replicating segment of DNA (a cloning vehicle or vector) that is able
to propagate in the host organism, and which can be linked to the DNA
segment to be cloned,
(iii) a procedure for introducing the composite DNA molecules into a
biologically functional recipient cell, and
(iv) a means of selecting those organisms that have acquired the desired
composite molecule.
In short, gene cloning or genetic engineering is essentially the insertion of a
specific piece of ‘foreign’ DNA into a cell, in such a way that the inserted DNA is
replicated and handed on to daughter cells during cell division. The main factors
involved in gene cloning are the following:
1. Isolation of the gene (or other piece of DNA) to be cloned.
2. Insertion of the gene into another piece of DNA called a vector, which will
allow it to be taken up by bacteria and replicated within them as the cells
grow and divide.
3. Transfer of the recombinant vectors into bacterial cells, either by
transformation or by infection using viruses.
4. Selection of those cells which contain the desired recombinant vectors.
5. Growth of the bacteria, that can be continued indefinitely, to give as much
cloned DNA as needed.
6. Expression of the gene to obtain the desired product.

2.1  Outline Of A Genetic Engineering Procedure

Genetic engineering requires two types of knowledge, namely, a grasp of the
concepts of molecular biology and a familiarity with laboratory manipulations.
The major steps in genetic engineering are briefly outlined in Figure 2.1.
(a) The first step is to break open living cells. A number of methods are
available to accomplish this. One popular way is to shear the cells in a
blender and then treat them with a detergent.
(b) The next step is to remove genetic information from cells. This is an easy
and straight-forward process; the information is stored in a chemical form
as part of DNA. Since DNA molecules are much longer than most other
large molecules found in cells, it has been possible to develop techniques
of purifying DNA. The DNA can be isolated and purified using different
techniques
 Genetic Engineering and Gene Cloning 2.3

Gene of interest

Human DNA

1 Break cell

Human cell
2 Remove DNA
3 Cut DNA

3a (a) From
cloning vehicle
3b
Cloning vehicle (b) From
(small, circular DNA) human cell

4 Mix DNAs
Gene of interest Recombinant
DNA molecule

Bacterial 5 Splice
DNA

6 Transfer DNA into bacterial cell

Bacterial cell
Engineered
bacterial cell

7 Engineered bacterial
cell divides manytimes

Figure 2.1 Major steps involved in gene cloning. 1. Human cells are broken (for clarity only
one cell is shown) 2. DNA containing the gene of interest is removed from human cells. 3. The
DNA from a cloning vehicle and human DNA are cut in specific places. The cloning vehicle DNA
is obtained from bacterial cells. 4. The two types of DNA are mixed. 5. The DNA fragments are
spliced together, yielding a recombinant DNA molecule. 6. The recombinant DNA molecule is
transferred into a bacterial cell, which has its own DNA. 7. The engineered cell created by step
6 is allowed to reproduce millions of times to form a clone. of identical cells. (Source: Drilica, K.
Understanding DNA and Gene Cloning. © 1984 New York, John Wiley and Sons Inc. Reprinted
by permission.)
 2.4 Biotechnology

(c) The third step is to cut away specific genes of interest from the rest of the
DNA. DNA is divided into segments which correspond to the letters in the
genetic code. When a number of segments or genetic letters are organized
in a specific combination, they create a gene. The molecular scissors used
to cut DNA into gene-sized pieces are called restriction endonucleases and
they recognize and cut at specific DNA sequences.
(d) The next step is to splice (join or incorporate) these specific sections of
DNA into agents called cloning vehicles (such as phages, plasmids etc.)
that carry the DNA sections into other living cells. Cloning vehicles are
relatively short DNA molecules that can penetrate the wall of a living
cell and can multiply inside that cell. The splicing process produces a
chimeric DNA molecule, containing a part of specific gene and a part of
cloning vehicle. Such a DNA molecule is also called a recombinant DNA
molecule. Once a foreign gene has been spliced into a cloning vehicle, both
the vehicle and the foreign gene are transferred (introduced) into a cell that
is normally a host for the vehicle. Usually the host cells are single-celled
organisms such as bacteria or yeast.
(e) The final step in gene cloning is to allow the host cell to multiply, forming a
clone having millions of identical cells. (In the example shown in the Figure
2.1 each member of the clone contains, in addition to its normal DNA
content, the same specific piece of human DNA joined to a cloning vehicle
DNA). By this process a piece of genetic information can be transferred
into a cell where it would never occur naturally. Hence a new organism can
be created.
In general, simply cloning a piece of DNA is not enough. The information in
DNA must be converted into a useful product. To make a product, the information
in DNA is usually transferred from the gene to the site where a new protein
molecule is manufactured through the process of gene expression.
Insulin, a controlling type of protein, serves as a good example to illustrate
one of the uses of recombinant DNA technology. The insulin gene is a region in
the DNA that contains information for producing insulin. Some diabetics fail to
produce sufficient quantities of insulin, and they are unable to properly control
their sugar metabolism; consequently, these patients must take daily injections of
insulin. Before the development of genetic engineering, insulin could be obtained
only by an expensive process of extracting the protein from hog (pig) pancreas,
but now, through gene cloning techniques, human insulin genes have been placed
in bacteria. Hog insulin is not advocated due to the following reasons:
(i) some people are allergic to it;
(ii) it is very expensive; and
(iii) it leads to the slaughtering of so many animals.
By genetic engineering, insulin is made inside bacteria. Thus, large quantities
of insulin are now produced by bacteria and it is easier to obtain insulin from
bacteria than from pancreas tissue. Moreover, the engineered bacteria produce
human insulin, an important feature for diabetics who have become allergic to
hog insulin.
 Genetic Engineering and Gene Cloning 2.5

To summarize, genetic engineering is a strategy for transferring small bits

of genetic information (DNA) from one organism to another. Certain pieces of
DNA will permanently alter the chemistry of the recipient organism in useful,
predictable and permanent ways.

2.2  Restriction Endonucleases

2.2.1  Nature
Enzymes that cut the phosphodiester bonds of polynucleotide chains are called
nuclease. Those nucleases that preferably break internal bonds are known as
endonuclease. During the 1970s, it was found that bacteria contained nucleases
that would recognize short nucleotide sequences with duplex DNA and cut the
phosphodiester backbone at highly specific sites on both strands of duplex. These
enzymes are called restriction endonucleases or simply restriction enzymes.
The discovery of a variety of restriction endonucleases is one of the reasons
for the rapid development of recombinant DNA. technology. Enzyme that cuts the
phosphodiester bonds of polynucleotide chains is called nuclease. The nuclease
that preferably breaks internal bonds is known as endonuclease. The enzyme
makes two incisions, one through each of the phosphate backbones of the double
helix without damaging the bases. Different endonucleases found in different
organisms recognize different nuclecotide sequences and therefore cut DNA at
different cleavage sites as depicted in Figure 2.2. Table 2.1 gives the list of some
restriction endonucleases and the site at. which they cleave DNA.
In nature, these restriction enzymes are used by the bacteria to destroy
various viral DNAs that might enter the cell, thereby restricting the potential
growth of the virus. Thus restriction enzymes serve as a defense mechanism. The
bacteria protect their own DNA from nucleolytic attack by ethylating the bases at
susceptible sites, a chemical modification that blocks the action of the enzyme.
Restriction enzymes are molecular scissors that are used to recognize and cut
DNA at specific sequences. The sites recognized by them are called recognition
sequences or recognition sites. By locating the positions of the cleavage sites
of a number of restriction enzymes in a DNA segment, restriction maps can be
prepared.
Sometimes restriction enzymes cleave both DNA strands at precisely
opposite points on the two strands, yielding blunt ended fragments. In some cases
the two DNA strands are not cut directly opposite each other. Instead, the cuts are
staggered forming cohesive ends(sticky ends). Figure 2.3 depicts the two types
of cleavages. Most restriction enzymes recognize only one short base sequence
in a DNA molecule and make two single-strand breaks, one in each strand,
generating 3¢-OH and 5¢-P groups at each position. The sequences recognized by
restriction enzymes are often palindromes—i.e. inverted repetitious sequences
which are symmetrical.
 2.6 Biotechnology

In a palindrome with rotational symmetry, the base sequence in the first half
of one strand of a DNA double helix is the mirror image of the second half of its
complementary strand. Restriction enzymes can cleave the DNA in three ways,
generating blunt ends, cohesive/ sticky ends with 3’ tails and cohesive/ sticky
ends with 5’ tails. The way of breaking the DNA solely depends on the restriction
enzyme. Each enzyme is named by a three letter (or four letter) abbreviation
(letters are italicized) that identifies its origin. Roman numerals (I, II, III) are
added to distinguish several enzymes with same origin (e.g., EcoRI).

Recognition sequenes
(a)
DNA

Cut site

Add restriction A A G C I T
endonuclease
to cut DNA G A A
T T C

Cut site

(b)
DNA
a b c d

A A G C T T
G A A
T T C Gently warm to
separate fragments

Figure 2.2 Cleavage of DNA by a Restriction Endonuclease. (a) A DNA molecule depicted
as two parallel lines, may contain many- short nucleotide sequences recognized by restriction
endonucleases. (b) When a restriction endonuclease is added to the DNA, it binds to the DNA
and cuts it. Some of these enzymes produce staggered cuts. The DNA molecule in the example
is converted into four shorter molecules, a, b, c, d each with staggered ends that can form base
pairs with each other. (Source: Drilica, K. Understanding DNA and Gene Cloning. © 1984 New
York, John Wiley and Sons Inc. Reprinted by permission.)
 Genetic Engineering and Gene Cloning 2.7

Table 2.1 Some restriction endonucleases, their source, recognition sequences and
sites of cleavage (indicated by arrow).

Enzyme Source Recognition sequence and

clearage site

Ø
Eco Rl Escherichia coli Ry13 G AATTC

GTTAA ØG

Ø
Hind II Haemophilus influenzae GTPy PuAC

CAPu ØPyTG

Ø
Hind III Haemophilus influenzae Rd A AGCTT

TTC GAØA

Ø
Hpa I Haemophilus parainfluenzae GTT AAC

CAAØTTG

Ø
Hpa II Haemophilus parainfluenzae CC GG

GGØCC

Ø
Bam HI Bacillus amyloliquefaciens G GATCC

CCTAGØG

Ø
Bgl III Bacillus globigi A GATCT

TCTAGØA

Ø
Hae II Haemophilus aegyptius PuGG GC Py

PyCGØCGPu

Ø
Hae III Haemophilus aegyptius GG CC

CCØGG

(Contd.)
 2.8 Biotechnology

Enzyme Source Recognition sequence and

clearage site

Ø
Hha I Haemophilus haemolyticus G CGC
CGC G
Ø

Ø
Pst I Providencia stuartii C TGCAG

GACGØTC

Ø
Sma I Serratia marcescens CCC GGG

GGGØCCC

Ø
Taq I Thermus aquaticus T CGA

AGCØT

Ø
Bal I Brevibacterium albidum TGG CCA

ACCØGGT

Ø
Sal I Streptomyces albus G G TCGAC

CAGCTØG

Ø
Xor II Xanthomonas oryzae C GAT CG

GCTAGØC

Ø
Alu I Arthrobacter luteus A GCT

TCGØA

Ø
Msp I Moraxella sp. CC GG

GGØCC

Ø
Mbo I Moraxella bovis G ATC
CTA G
Ø

Ø
Sau 3 Al Staphylococcus aureus 3A GA TC
CT AG
≠
 Genetic Engineering and Gene Cloning 2.9

(a) Cuts on line (b) Cuts symmetrically placed

of symmetry around line of symmetry

. . . TC GA. . . . . . GAA TTC. . .

. . . AG CT. . . . . . CTT AAG. . .

Separation of Separation of
fragments fragments

3¢ 5¢ 3¢ 5¢
. . . TC + GA. . . . . .G + AATTC. . .
. . . AG CT. . . CTTAA. . . G. . .
5¢ 3¢ 5¢ 3¢
Blunt-end molecules Cohesive molecules

Figure 2.3 Two types of cuts made by restriction enzymes. The arrows indicate the cleavage
sites. The dashed line is the centre of symmetry of the sequence. (Source: Hartl, D.L.; Freifelder,
D.; Snyder, I.A. Basic Genetics. © 1988 Boston: Jones and Bartlett Publishers. Reprinted by
permission.)

2.2.2 Properties
Many restriction enzymes have been isolated so far. All of these enzymes have
been found in prokaryotes; no similar enzymes have been identified in the few
eukaryotic organisms that have been examined.
The restriction enzymes fall into three types designated as Type I, Type II
and Type III. In Type I and III, both the methylase and restriction activities are
carried out by a single large enzyme complex. In Type II, the restriction enzyme
is independent of its methylase and cleavage occurs at very specific sites that
are within or close to the recognition sequence. Some enzymes recognize a
specific nucleotide pair sequence and then cleave the DNA at a non-specific site
away from that recognition site. Some enzymes cleave the DNA at the specific
recognition site. All the enzymes are sequence specific, and thus the number
of cuts they make in a particular DNA molecule or population of molecules is
dependent upon the number of times the particular sequence is present in the
DNA.
The sequences recognized by restriction enzymes are 4 to 8 nucleotides
long and characterized by a particular type of internal symmetry. Besides
cleavage, modification in the form of methylation is also brought about by some
enzymes. They are called modification enzymes (methylases). This methylation
distinguishes genes in different states of functioning. There are also enzymes
which perform the function of restriction and modification. Based on these
attributes restriction enzymes have been grouped into different types: type II
restriction enzymes systems (e.g. EcoRI), which have different enzymes for
modification and restriction; type I (EcoK) and type III (EcoPI) enzyme systems,
in which the same enzyme possesses both activities although the restriction and
modification sites differ in position.
 2.10 Biotechnology

Of the above two classes of restriction enzymes, type II are most important
for cloning purposes. Enzymes with 4 bp target sites are used when frequent cuts
are desired to get small DNA fragments and those with 8 bp are used when rare
cuts are desired to get long DNA segments. Otherwise majority of the enzymes
have 6 bp target sites. Some of them can cleave both methylated as well as
unmethylated targets, but majority of them cleave only unmethylated targets.
One of the most important events in the study of restriction enzymes was the
observation by electron microscope that fragments produced by many restriction
enzymes spontaneously circularize. Figure 2.4 depicts this phenomenon. The
circles could be relinearized by heating, but if after circularization they were also
treated with E. coli DNA ligase, which joins 3¢-OH and 5¢-P groups, circularization
became permanent. The Eco Rl enzyme, discovered in the laboratories of Dussoix
and Boyer, was also able to cleave a circular DNA molecule to form a linear
duplex with complementary ends. Figure 2.5 illustrates this.

TGCA TGCA TGCA

ACGT ACGT ACGT

T GCA T GCA T GCA

ACG T ACG T ACG T

C G
A TGCA T
TGCA

Figure 2.4 Circularization of DNA fragments produced by a restriction enzyme. Arrows indicate
cleavage sites. (Source: Hartl, D.L.; Freifelder, D.; Snyder, L.A. Basic Genetics. © 1988 Boston:
Jones and Bartlett Publishers. Reprinted by permission.)

Most restriction enzymes recognize one base sequence without regard to the
source of the DNA. Thus, fragments obtained from a DNA molecule from one
organism have the same cohesive ends as the fragments produced by the same
enzyme acting on DNA molecules from another organism. This property is one
of the foundations of the recombinant DNA technology. Since most restriction
enzymes recognize a unique sequence, the number of cuts made in the DNA
from an organism, by a particular enzyme is limited. Of special interest are the
smaller DNA molecules, such as viral or plasmid DNA, which may have only
1–10 sites of cutting (or even none) for particular enzymes. Plasmids having a
single site for a particular enzyme are especially valuable.
 Genetic Engineering and Gene Cloning 2.11

Circular DNA molecule

Axis of
symmetry

G AA T T C
C T T AA G

Eco R1 cleavage

G AATTC
CTTAA G

Figure 2.5 A circular DNA molecule with a single recognition site for Eco Rl restriction enzyme
is cleaved to a linear duplex with complementary ends. Note the symmetry in recognition site and
cleavage pattern

Because of the sequence specificity, a particular restriction enzyme generates

a unique set of fragments for a particular DNA molecule. Another enzyme will
generate a different set of fragments from the same DNA molecule. Figure 2.6
shows the sites of cutting of E. coli phage lambda DNA by the enzymes Eco R1
and Bam HI. A map showing the unique sites of cutting of the DNA of a particular
organism by a single enzyme is called a restriction map. The family of fragments
generated by a single enzyme can be detected easily by gel electrophoresis of
enzyme-treated DNA and particular DNA fragments can be isolated by cutting
out the portion of the gel containing the fragment and removing the DNA from
the gel.
0 43.9 53.5 64.9 79.9 91.8 100
43.9 9.6 11.4 15.0 11.9 8.2

0 10.6 45.3 56.5 69.3 83.8 100

10.6 34.7 11.2 12.8 14.5 16.2

Figure 2.6 Restriction maps of a DNA for the restriction enzymes Eco Rl and Bam Hl. The
vertical bars indicate the sites of cutting. The numbers above the line indicate the percentage
of the total length of DNA measured from the end of the molecule arbitrarily designated as
the left end. The numbers below the line are the length of each fragment, again expressed as
percentage of the total length. (Source: Hartl, D.L.; Freifelder, D.; Snyder, L.A. Basic Genetics. ©
1988 Boston: Jones and Bartlett Publishers. Reprinted by permission.)

Several techniques are used to locate particular genes on fragments of

a restriction map. One of the most common procedures is Southern blotting.
In Southern blotting, a gel in which DNA molecules have been separated by
 2.12 Biotechnology

electrophoresis is treated with alkali to render the DNA single stranded (denature
the DNA) and then the strand is transferred to a sheet of nitrocellulose so that the
relative positions of the DNA bands are maintained. Figure 2.7 illustrates this. The
nitrocellulose, to which the single-stranded DNA tightly binds, is then exposed
to radioactive RNA or DNA probe which leads to renaturation. Radio labeled
probe becomes stably bound (resistant to removal by washing) to the DNA only
at positions at which base sequences complementary to the radio labeled probes
are present. The radioactivity is located by placing the paper in contact with
X-ray film; after development of the film, blackened regions indicate positions of
radioactivity. If a radioactive mRNA species transcribed from a particular gene
is used (e.g., mRNA isolated from a specialized cell that predominantly makes
one type of RNA), it will hybridize only with the restriction fragment containing
that gene.

Absorbent paper stack

Nitrocellulose
Gel Buffer

Figure 2.7 Southern blotting. Due to capillary action of absorbent paper stack, buffer travels
through gel into absorbent paper stack. The DNA fragments will also be carried from the gel
along with the buffer, which can’t cross the membrane and adhere to it.

2.3  Cloning Vehicles or Vectors

Cloning vehicles are small plasmid, phage, or animal virus DNA molecules used
to transfer a DNA fragment from a test tube into a living cell. Cloning vehicles
are also called vectors. Vectors have the ability to replicate by themselves. There
can be cloning vectors and expression vectors. Cloning vectors are used for
multiplying DNA inserts in a suitable host. Transcription or translation of cloned
gene does not occur. Cloning vectors are used in creating genomic library or
gene bank, in the preparation of probe etc. Expression vector is used to express
the cloned gene, i.e. transcription and translation of the cloned gene occurs,
due to which a protein is produced. Expression vectors are used in producing
transgenic plants or animals.
To be useful, a vector must have the following properties:
1. It should have the ability of self-replication so that many copies of the
DNA insert can be formed as the vector replicates.
2. It should get introduced into the host cell very easily.
3. It should contain unique target sites for restriction enzymes so that foreign
DNA can be inserted without disrupting its function
4. It should have promoter, operator etc., if the expression of foreign DNA is
to be verified.
 Genetic Engineering and Gene Cloning 2.13

One of the most important uses of recombinant DNA technology is the

cloning of (i) random DNA or cDNA segments, often used as probes or (ii)
specific genes, which may be either isolated from the genome or synthesized
either organochemically or in the form of cDNA from mRNA. This cloning of
DNA is possible only with the help of another DNA molecule, which is capable
of replicating in the host. This other DNA molecule is often used in the form of a
vector, which could be a plasmid, a bacteriophage, a derived cosmid, a phagemid
(phage + plasmid) or even a virus. Sometimes vectors are modified by inserting
a DNA segment to locate unique site(s) for one or more restriction enzymes to
facilitate its use in gene cloning. This inserted DNA is sometimes called ‘poly
linker’.
Certain types of small DNA molecules are infectious. Once inside a living
cell, they can utilize the machinery of the cell for reproduction. According to the
biochemist’s definition of life, these DNA molecules are not alive; they cannot
reproduce by themselves. Among other things they need RNA polymerase from
their host (the infected bacterium) to transcribe their DNA into messenger RNAs.
They also need host ribosomes to translate the messages into proteins. But even
with these deficiencies, infectious DNAs can have profound effects on living
cells. Some types take over a cell and kill it, while other types can be beneficial
to a cell.
These infectious DNA molecules fall into two general types, the viruses
and the plasmids. Viruses surround their DNA molecules with a protective shell
of protein; thus, they can sometimes survive for many years outside their host
cell. Plasmids, on the other hand, are naked, circular DNA molecules, generally
found inside cells only. It is possible to cut DNA molecules from plasmids and
bacteriophages (viruses that attack bacteria) at a specific place, insert a piece
of DNA from another source, and still retain all the information necessary for
infection by the plasmid or phage. Thus these infectious DNA molecules are
useful as tools to transfer DNA from one type of cell to another.

2.3.1 Plasmids
Plasmids are small, circular, double-stranded, autonomously replicating,
covalently bonded DNA molecules (Fig. 2.8) that occur naturally in bacteria. Like
all natural DNA molecules, plasmids contain a special region in their DNA called
an origin of replication. The origin serves as a start signal for DNA polymerase
and ensures that the plasmid DNA molecule will be replicated by the host cell.
Many kinds of plasmids have been discovered. Plasmids differ in length
and in the genes contained in their DNA. Some of the smaller plasmids which
are popular in gene cloning, have about 5000 nucleotide pairs, enough DNA
to code for about five average-sized proteins. In comparison, E. coli contains
slightly more than four million nucleotide pairs in its DNA, and we have about
four billion nucleotide pairs in our DNA. There are plasmids which are larger
and are difficult to handle (e.g. transmissible plasmids). They are generally not
used in gene cloning. Some plasmids have the ability to integrate into the host’s
 2.14 Biotechnology

chromosome, and these are called episomes. The F factor that is involved with
conjugation of E. coli is an example of an episome.

(a)Col EI type DNA molecules (b) Enlargement of plasmid similar to CoI EI

Figure 2.8 Magnified plasmids as observed under electron microscope (diagramatic) (a) Four
DNA molecules of the type called Col El. These small, circular DNAs are only 0.001 times the
length of E. coli DNA. (b) Enlargement of plasmid similar to col El showing a small portion with
separation of strand. (Source: Drilica, K. Understanding DNA and Gene Cloning. © 1984 New
York, john Wiley and Sons Inc. Reprinted by permission.)

An important aspect of plasmid DNA molecules is that they often contain

genes that make their host bacterial cell resistant to antibiotics. These plasmids
carry antibiotic resistant genes that play an important role in the identification
and selection of recombinant DNA molecules. In general the plasmid vehicles
have different buoyant density than that of the host DNA and thus they can be
easily purified. A plasmid that has been used extensively for molecular cloning is
pBR 322 (Fig. 2.9). This plasmid is of the non-conjugal type; it will not promote
conjugation, and each E. coli cell transformed with it will have six to eight
copies per host chromosome. pBR 322 is a plasmid that has been engineered in
the laboratory from natural plasmids so that it has features which are useful for
molecular cloning experiments. Its replication in the E. coli cell is dependent
upon the presence of rep. the origin of replication sequence. pBR 322 is 4363
base pairs long, weighing 2.7 ¥ 106 daltons.
Another series of plasmids that are used as cloning vectors belong to pUC
series. They are available in pairs with reversed orders of restriction sites relative
to lacZ promoter. pUC8 and pUC9 make one pair. These plasmid vectors are
often used for cloning DNA segments of small size (upto 10 kilobases).
pSC 101 derived from plasmid R-6-5 is the first cloning vehicle to be
described. It has a single Eco R1 substrate site. It specifies tetracycline resistance
and can replicate autonomously. Insertion of foreign DNA into this plasmid does
not alter its other functions, specially those of resistance and replication. This has
been successfully used for the cloning of antibiotic resistant genes, ribosomal
DNA from the toad Xenopus and histone genes from the sea-urchin. The only
disadvantage with pSC 101 is that the wild type cannot be distinguished from the
recombinant derivatives.
This is a tumour inducing plasmid carried by the bacterium Agrobacterium
tumefaciens. The bacterium transfers the plasmid into plant cells where it
 Genetic Engineering and Gene Cloning 2.15

induces the formation of crown galls. Modified forms of the Ti plasmid are now
being used for genetic engineering of plants. More details are given in chapter 4.
pUC 18 is a popular and widely used plasmid first prepared in the University
of California. It has 2686 bp, ampicillin resistant gene and Lac Z gene. It has
multiple cloning sites.
Several plasmids were found to carry genetic factors for fertility, resistance
to antibiotics, ability to ferment sugars and even hydrocarbons like petroleum,
and for the production of bacteriocins and haemolysins. All these kinds of
plasmids have duly been altered to yield smaller plasmids suitable as small
vehicles. They are classified on the basis of their compatibility characteristics.
Plasmids are considered incompatible when they fail to establish themselves due
to competition for essential replication sites on the cell membrane.
Eco R1

Eco R1 Sal 1
Pst 1

Ampicillin Sal 1
resistance Pst 1

+
Tetracycline
resistance

Origin of replication

Plasmid pBR322 and its major characteristic

pUC-8 pUC-8
(2768 bp) Sma I (2768 bp)
Bam Eco RI Sma I
Sal I HI Eco RI
Pst I Bam HI
Hind III
Sal I
Pst I
Hind III

Operator
Hae II Promoter

b Galactosidase Hae II
gene
pUC

Ampicillin
resistance

Plasmid pUC and its major characteristic feature

Figure 2.9 Physical map of the pBR 322 and pUC cloning vehicles
 2.16 Biotechnology

Some plasmids belonging to group P exhibit promiscuity and are able to infect
a wide range of gram-negative bacteria. Therefore they are used to introduce
new genes. F plasmids are naturally occurring sex-factor plasmids which induce
conjugation. These are not used in cloning experiments.
The F plasmid integrates itself into the E. coli chromosome randomly and
occasionally exhibits excision along with a portion of bacterial material and
thus is capable of performing recombinant function. Phages are also known to
integrate themselves with F particles and acquire a transducing function.
R plasmids carry genes for antibiotic resistance and several of them have
a wide host range. For instance, RP4, originally isolated from Pseudomonas, is
easily transferred by conjugation to most gram-negative bacteria including E.
coli. These R plasmids have two unique segments one carrying the resistance
determinant (r-det) and the other resistance transfer factor (R-tf).
5. Col plasmids (colicin plasmids) are those which code for bacterial
antibodies called colicin. For example, col. E1 specifies the production of the
substance colicin E1 and exists in multiple copies in a cell (20-30 copies).
In the presence of chloramphenicol, it gets amplified, i.e. while the bacterial
chromosome stops replicating, col. El continues to replicate for 12–16 hours,
giving rise to 1000–3000 copies per cell, constituting approximately 45% of the
total cellular DNA. Col. E1 has a single Eco R1 site and once foreign DNA
is inserted it no longer produces colicin and therefore recombinant plasmids
are easily identifiable. Some plasmids have been constructed which contain
a fragment of lambda DNA including the cos site. These plasmids have been
termed cosmids and can be used as vectors. Cosmids are essentially plasmids
having about 250 bp of λ phage DNA. Cosmid vector is made up of pBR 322
and λ segment with cos sites, a Pst1 and Pvu1 recognition site for restriction
enzymes within gene ampicillin resistance and Bam H1. Some viruses are used
to develop the vectors for animal cells (See Chapter 7).

2.3.2 Bacteriophages
Bacteriophages are viruses which infect bacteria. These are commonly called
phages. They are more complicated than plasmids. In addition to having an
origin of replication, phage DNA contains genes coding for proteins that form a
protective shell around the DNA. But, like plasmids, phages lack the machinery
necessary to actually make proteins; consequently, they reproduce only inside
living bacterial cells.
Based on their nucleic acid composition, they are further grouped as double
stranded DNA (dsDNA) single stranded DNA (ssDNA), dsRNA and ssRNA
phages. Virus enzymes like lysozyme are made by the phages to get out of the
host. These enzymes degrade peptidoglycan.
Many phages are like miniature hypodermic syringes (Fig. 2.10). The phage
DNA is wrapped into a tight ball inside a head-like structure made of protein. A
tail, also made of proteins, is attached to the head. When such a phage particle
comes into contact with a bacterial cell, the phage tail sticks to the cell wall, and
 Genetic Engineering and Gene Cloning 2.17

the DNA is squirted out of the head, through the tail, and into the bacterium. Soon
after the phage DNA gets into the cell, it begins to take control. Special phage genes
are transcribed by the bacterial RNA polymerase, and the resulting messenger
RNAs are translated into phage proteins using the bacterial ribosomes. At early
stages of infection some phages produce proteins that destroy the bacterial DNA,
chopping it into individual nucleotides. Once that has happened, the bacterium
is doomed, because all the information needed for its own reproduction is gone.
Some phages have genes that produce an RNA polymerase, so they do not have
to rely on the host polymerase to make messenger RNA from phage genes. The
phage lambda permits cloning of segments up to 20-25 kb long and cosmid or
phagmid vectors permit cloning of segments up to 45 kb long.
Phage efficiency is awesome. One phage produces hundreds of progeny
particles. Each progeny particle can infect a bacterial cell and produce several
hundred more phage particles. By repeating the infection cycle just four times, a
single phage particle can lead to the death of more than one billion bacterial cells.
If a DNA fragment is spliced into a phage DNA molecule without destroying
important phage genes, the phage will reproduce the fragment along with its own
DNA when it infects a bacterial cell.

a b

c d

Figure 2.10 Magnified bacteriophages as observed under electron microscope.

(a) Bacteriophage P2, magnification 266,000 times. (b) Bacteriophage lambda, magnification
109,000 times (c) Bacteriophage T5, magnification 91,000 times. (d) Bacteriophage T4,
magnification 1,80,000 times. (Source: Drilica, K. Understanding DNA and Gene Cloning. © 1984
New York, John Wiley and Sons Inc. Reprinted by permission.)
 2.18 Biotechnology

Lambda Phages (Temperate bacteriophage)

One of the phages used for cloning is called lambda. Lambda is slightly more
sophisticated than other phages. When lambda DNA is injected into a bacterial
cell, it has two choices. It can multiply and destroy the bacterium, or it can
take up residence in the cell. This is referred to as a lysogenic cycle. When the
latter choice is made the lambda DNA inserts into the bacterial chromosome;
it becomes part of the bacterial chromosome (Fig. 2.11). The phage genes that
normally would produce the proteins to kill the cell are turned off by a repressor
protein made from phage gene. Thus every time the bacterial DNA replicates,
lambda replicates. In this dormant state lambda DNA does a little more than
produce repressor protein to keep its genes shut down. At the same time, the
repressor protects the bacterial cell from infection by other lambda phages; when
the newcomers inject their DNA, it is soon bound by a repressor produced from
the resident lambda DNA. Thus the incoming DNA is unable to initiate a lytic
infection that would kill the cell. Consequently, one can easily find lysogens
(bacterial cells that are being protected by a resident phage).

1 2

Phage DNA Bacterial DNA

5 4

Figure 2.11 Formation of a lysogen. (1) Bacteriophage lambda injects DNA through the
bacterial cell wall. The resulting linear DNA molecule has sticky ends. (2) The DNA circularizes
and (3) becomes ligated. At this point the phage has two choices. It can replicate its DNA,
produce progeny phage, and kill the bacterium. Alternatively, it can integrate its DNA into the
bacterial DNA (4-6) and remain quiescent for an indefinite number of bacterial generations. Both
phage and bacterial proteins play important roles in the integration process. (Source: Drilica, K.
Understanding DNA and Gene Cloning. © 1984 New York, John Wiley and Sons Inc. Reprinted
by permission.)
Exploring the Variety of Random
Documents with Different Content
“Never beat about the bush where you deal with Jake Shackleton,”
she said, slipping her hand in Mariposa’s arm as they passed down
the corridor. “He’s got no use for people who gambol round the
subject. Say your say and then go. That’s the way to get on with
him.”
In the anteroom the boy was still sitting, his chair tilted back on its
hind legs, The Trumpet in his hands. Nevertheless, he had made an
incursion into the inner regions to find out whom Mrs. Willers was
piloting into the sanctum, for he had the curiosity of those who hang
on the fringes of the newspaper world.
As the ladies passed him, going toward the stair-head, a young man
rose above it, almost colliding with them. Then in the gloom of the
dejected gas-jets he stood aside, against the wall, letting them pass
out. He wore a long ulster with a turned-up collar. Between the edge
of this and the brim of his derby hat, there was the gleam of a pair
of eye-glasses and a suggestion of a fair mustache. He raised his
hat, holding it above his head during the interval of their transit,
disclosing a small pate clothed with smooth blond hair.
“Who was that lady with Mrs. Willers?” he said to the boy, as he
walked toward the door into the corridor.
“She’s some singing lady,” answered that youth drawlingly, tilting his
chair still farther back, “what’s come to see Mr. Shackleton about
singing at the opera-house. Her name’s Moreau.”
The young man, without further comment, passed into the inner
hall, leaving the boy smiling with pride that his carelessly-acquired
information should have been so soon of use. For the questioner
was Winslow Shackleton, the millionaire’s only son.
The next morning was one of feverish excitement in the cottage on
Pine Street. Mariposa could not settle herself to anything, at one
moment trying her voice at the piano, at the next standing in front
of her glass and putting on all her own and her mother’s hats in an
effort to see in which she presented the most attractive appearance.
She thrilled with hope for a space, then sank into a dead apathy of
dejection. Lucy was quietly encouraging, but the day was one of
hidden anguish to her. The daughter, ignorant of the knowledge and
the memories that were wringing the mother’s heart, wondered why
Lucy was so confident of her winning Shackleton’s approval. As the
hour came for her to go she wondered, too, at the marble pallor of
her mother’s face, at the coldness of the hand that clung to hers in a
lingering farewell. Lucy was giving back her child to the father who
had deserted it and her.
The excitement of the morning reached its climax when a carriage
appeared at the curb with Mrs. Willers’ face at the window. The hour
of fate had struck, and Mariposa, with a last kiss to her mother, ran
down the steps feeling like one about to embark on a journey upon
perilous seas in which lie enchanted islands.
During the drive Mrs. Willers talked on outside matters. She was
business-like and quiet to-day. Even her clothes seemed to partake
of her practical mood and were inconspicuous and subdued. As the
carriage turned down Mission Street she herself began to experience
qualms. What if they had all been mistaken and the girl’s voice was
nothing out of the ordinary? What a cruel disappointment, and with
that sick, helpless mother! What she said was:
“Now, here we are! Remember that you’ve got the finest voice
Lepine’s ever likely to hear, and you’re going to sing your best.”
They alighted, and as they turned into the flagged entrance that led
to the foyer, Shackleton came forward to meet them. He looked
older in the crude afternoon light, his face showing the lines that his
fiercely-lived life had plowed in it. But he smiled reassuringly at
Mariposa and pressed her hand.
“Everything’s all ready,” he said; “Lepine’s put back a rehearsal for
us, so we mustn’t keep him waiting. And are you all ready to
surprise us?” he asked, as they walked together toward where the
three steps led to the foyer.
“I’m ready to do my best,” she answered; “a person can’t do more
than that.”
The answer pleased him, as everything she said did. He saw she was
nervous, but that she was going to conquer herself.
“Lots of grit,” he said to himself as he gave ear to a remark of Mrs.
Willers’. “She won’t quit at the first obstacle.”
They passed through the opening in the brass rail that led to the
foyer. This space, the gathering place of the radiant beings of
Mariposa’s first night at the opera, was now a dimly-lit and deserted
hall, its flagged flooring looking dirty in the raw light. From
somewhere, in what seemed a far, dreamy distance, the sound of a
piano came, as if muffled by numerous doors. As they crossed the
foyer toward the entrance into the auditorium, the door swung open
and two men appeared.
One was a short and stout Frenchman, with a turned-over collar,
upon which a double chin rested. He had a bald forehead and eyes
that gleamed sharply from behind a pince-nez. At sight of the trio,
he gave an exclamation and came forward.
“Our young lady?” he said to Mariposa, giving her a quick look of
scrutiny that seemed to take her in from foot to forehead. Then he
greeted Shackleton with slightly exaggerated foreign effusion. He
spoke English perfectly, but with the inevitable accent. This was
Lepine, the impresario, and the other man, an Italian who spoke
little English, was presented as Signor Tojetti, the conductor.
They moved forward talking, and then, pushing the door open,
Lepine motioned Mariposa to enter. She did so and for a moment
stood amazed, staring into a vast, shadowy space, where, in what
seemed a vague, undefined distance, a tiny spot or two of light cut
into the darkness. The air was chill and smelt of a stable. From
somewhere she heard the sound of voices rising and falling, and
then again the notes of a piano, now near and unobscured,
carelessly touched and resembling, in the echoing hollow
spaciousness of the great building, the thin, tinkling sounds emitted
by smitten glass.
Lepine brushed past her and led the way down the aisle. As she
followed him her eyes became accustomed to the dimness, and she
began to make out the arch of the stage with blackness beyond, into
which cut the circles of light of a few gas-jets. The lines of seats
stretched before her spectral in linen covers. Now and then a figure
crossed the stage, and as they drew nearer, she saw on one side of
it a man sitting on a high stool reading a paper book by the light of
a shaded lamp. The notes of the piano sounded sharper and closer,
and by their proximity more than by her sight, she located it in a
dark corner of the orchestra. As they approached, the sound of two
voices came from this corner, then suddenly a man’s smothered
laugh.
“Mr. Martinez,” said Lepine, directing his voice toward the darkness
whence the laugh had risen, “the lady is here to sing, if you are
ready.”
Instantly a faintly luminous spark, Mariposa had noticed, bloomed
into the full-blown radiance of a gas-jet turned full cock under a
sheltering shade. It projected, what seemed in the dimness, a
torrent of light on the keyboard of the piano, illuminating a pair of
long masculine hands that had been moving over the keys in the
darkness. Behind them the girl saw a shadowy shape, and then a
spectacled face under a mane of drooping black hair was advanced
into the light.
“Has the lady her music?” said the face, in English, but with another
variety of accent.
She handed him the two songs she had brought, “Knowest thou the
Land,” from Mignon, and “Farewell, Lochaber.” In the short period of
her tuition her teacher had told her that she had sung “Lochaber”
admirably. The man opened them, glanced at the names, and
placing the “Mignon” aria on the rack, ran his hands lightly and
carelessly over the keys in the opening bars of the accompaniment.
“Whenever the lady is ready,” he said, with an air of patience, as
though he had endured this form of persecution until all spirit of
revolt was crushed.
Mariposa drew back from him, wondering if she were to sing there
and then. Lepine was behind her, and behind him she saw, with a
sense of nostalgic loneliness, that the Italian conductor was
shepherding Mrs. Willers and Shackleton into two seats on the aisle.
They looked small and far away.
“We will mount to the stage this way, Mademoiselle,” said Lepine,
and he indicated a small flight of steps that rose from the corner of
the orchestra to the lip of the stage above.
He ascended first, she close at his heels, and in a moment found
herself on the dark, deserted stage. It seemed enormous to her,
stretching back into unseen regions where the half-defined shapes of
trees and castles, walls and benches were huddled in dim confusion.
Down the aisles between side-scenes she caught glimpses of vistas
lit by wavering gleams of light. People moved here and there, across
these vistas, their footsteps sounding singularly distinct. As she
stood uneasily, looking to the right and left, a sudden sound of
hammering arose from somewhere behind, loud and vibrant. Lepine,
who was about to descend the stairs, turned and shouted a furious
sentence in Italian down the opening. The hammering instantly
ceased, and a man in white overalls came and stared at the stage.
The impresario, charily—being short and fat—descended the stairs.
“Now, Mademoiselle,” he said, speaking from the orchestra, “if you
are ready, come forward a little, nearer the footlights there.”
Mariposa moved forward. Her heart was beating in her throat, and
she felt a sick terror at the thought of what her voice would be like
in that huge void space. She was aware that the man who had been
reading the paper book had closed it and was leaning his elbow on
the lamp-stand, watching her. She was also aware that a woman and
a man had suddenly appeared in the lower proscenium box close
beside her. She saw the woman dimly, a fat, short figure in a light-
colored ulster. Whispering to the man, she drew one of the linen-
covered chairs close to the railing and seated herself.
“Is the lady ready?” said the pianist, from his dark corner.
“Quite ready,” replied Mariposa, hearing her voice like a tremulous
thread of sound in the stillness.
The first bars of the accompaniment sounded thinly. Mariposa
stepped forward. She could see in the shadowy emptiness of the
auditorium Lepine’s bald head where he sat alone, half-way up the
house, and the two pale faces of Shackleton and Mrs. Willers. The
Italian conductor had left them and was sitting by himself at one
side of the parquet. In the stillness, the notes of the piano were
curiously tinkling and feeble.
Mariposa raised her chest with a deep inspiration. A sudden excited
expectation seized her at the thought of letting her voice swell out
into the hushed void before her. The listening people seemed so
small and insignificant in it, they suddenly lost their terror. She
began to sing.
It seemed to her that her first notes were hardly audible. They
seemed as ineffectual as the piano. Then her confidence grew, and
delight with it. She never before had felt as if she had enough room.
Her voice rolled itself out like a breaking wave, lapping the walls of
the building.
The first verse came to an end. The accompaniment ceased. Lepine
moved in his distant seat.
“Continue, Mademoiselle,” he said sharply; “the second verse, if you
please. Again, Mr. Martinez.”
Mariposa saw the woman in the box look at the man beside her,
raise her eyebrows, and nod.
She began the second verse and sang it through. As its last notes
died out there was silence for a moment. In the silence the Italian
conductor rose and came forward to where Lepine sat. Mariposa,
standing on the stage, saw them conferring for a space. The Italian
talked in a low voice, with much gesticulation. Shackleton and Mrs.
Willers were motionless and dumb. The woman in the box began to
whisper with the man.
“And now the second piece, if Mademoiselle has no objection,” came
the voice of the impresario across the parquet. “One can not judge
well from one song.”
The second song, “Lochaber,” had been chosen by Mariposa’s
teacher to show off her lower register—those curious, disturbing
notes that were so deep and full of vague melancholy. She had
gained such control as she had over her voice and sang with an
almost joyous exultation. She had never realized what it was to sing
before people who knew and who listened in this way in a place that
was large enough.
When the last notes died away, the tinkling of the piano sounding
like the frail specters of music gafte the tones of the rich, vibrant
voice, there was a sudden noise of clapping hands. It came from the
box on the right, where the woman in the ulster was leaning over
the rail, clapping with her bare hands held far out.
“Brava!” she cried in a loud, full voice. “Brava! La belle voix! Et quel
volume! Brava!”
She bounced round on her chair to look at the man beside her, and,
leaning forward, clapped again, crying her gay “brava.”
Mariposa walked toward the box, feeling suddenly shy. As she drew
nearer she saw the woman’s face more distinctly. It was a dark
French face, with a brunette skin warming to brick-dust red on the
cheeks, set in a frame of wiry black hair, and with a big mouth that,
laughing, showed strong white teeth, well separated. As Mariposa
saw it fairly in the light of an adjacent lamp she recognized it as that
of the Leonora of “Il Trovatore.” It was the prima donna.
She started forward with flushing cheek and held out a hesitating
hand. The fat, ungloved palms of the singer closed on it with Gælic
effusion. Mariposa was aware of something delightfully wholesome
and kind in the broad, ruddy visage, with its big, smiling mouth and
the firm teeth like the halves of cleanly-broken hazelnuts. The singer,
leaning over the rail, poured a rumbling volume of French into the
girl’s blushing, upturned face. Mariposa understood it and was trying
to answer in her halting schoolgirl phrases, when the voice of Mrs.
Willers, at the bottom of the steps, summoned her.
“Come down, quick! They think it’s fine. Oh, dearie,” stretching up a
helping hand as Mariposa swept her skirts over the line of the
footlights, “you did fine. It was great. You’ve just outdone yourself.
And you looked stunning, too. I only wished the place had been full.
Heavens! but I thought I’d die at first. While you were standing
there waiting to begin I felt seasick. It was an awful moment. And
you looked just as cool! Mr. Shackleton don’t say much, but I know
he’s tickled to death.”
They walked up the aisle as she talked to where Shackleton and the
two men were standing in earnest conversation. As they approached
Lepine turned toward her and gave a slight smile.
“We were saying, Mademoiselle,” he said, “that you have
unquestionably a voice. The lower register is remarkably fine. Of
course, it is very untrained; absolutely in the rough. But Signor
Tojetti, here, finds that a strong point in your favor.”
“Signor Tojetti,” said Shackleton, “seems to think that two years of
study would be ample to fit you for the operatic stage.”
Mariposa looked from one to the other with beaming eyes, hardly
able to believe it all.
“You really did like it, then?” she said to Lepine with her most
ingenuous air.
He shrugged his shoulders, with a queer French expression of
quizzical amusement.
“It was a truly interesting performance, and after a period of study
with a good master it should be a truly delightful one.”
The Italian, to whom these sentences were only half intelligible, now
broke in with a quick series of sonorous phrases, directed to Lepine,
but now and then turned upon Shackleton. Mariposa’s eyes went
from one to the other in an effort to understand. The impresario,
listening with frowning intentness, responded with a nod and a word
of brusk acquiescence. Turning to Shackleton, he said:
“Tojetti also thinks that the appearance of Mademoiselle is much in
her favor. She has an admirable stage presence”—he looked at
Mariposa as if she were a piece of furniture he was appraising. “Her
height alone is of inestimable value. She would have at least five
feet eight or nine inches.”
At this moment the lady in the box, who had risen to her feet, and
was leaning against the railing, called suddenly:
“Lepine, vraiment une belle voix, et aussi une belle fille! Vous avez
fait une trouvaille.”
Lepine wheeled round to his star, who in the shadowy light stood, a
pale-colored, burly figure, buttoning her ulster over her redundant
chest.
“A moment,” he said, apologetically to the others, and, running to
the box, stood with his head back, talking to her, while the prima
donna leaned over and a rapid interchange of French sentences
passed between them.
Signor Tojetti turned to Mariposa, and, with solemn effort, produced
an English phrase:
“Eet ees time to went.” Then he waved his hand toward the stage.
The sound of feet echoed therefrom, and as Mariposa looked, an
irruption of vague, spectral shapes rose from some unseen
cavernous entrance and peopled the orchestra.
“It’s the rehearsal,” she said. “We must be going.”
They moved forward toward the entrance, the auditorium behind
them beginning to resound with the noise of the incoming
performers. A scraping of strings came from the darkened orchestra,
and mingled with the tentative chords struck from the piano. At the
door Lepine joined them, falling into step beside Shackleton and
conversing with him in low tones. Signor Tojetti escorted them to the
brass rail and there withdrew with low bows. The ladies made out
that the rehearsal demanded his presence.
Once again in the gray light of the afternoon they stood for a
moment at the curb waiting for the carriage.
Lepine offered his farewells to Mariposa and his wishes to see her
again.
“In Paris,” he said, giving his little quizzical smile—“that is the place
in which I should like to see Mademoiselle.”
“We’ll talk about that again,” said Shackleton; “I’m going to see Mr.
Lepine before he goes and have another talk about you. You see,
you’re becoming a very important young lady.”
The carriage rolled up and Mariposa was assisted in, several street
boys watching her with wide-eyed interest as evidently a personage
of distinction.
Her face at the window smiled a radiant farewell at the group on the
sidewalk; then she sank back breathless. What an afternoon! Would
the carriage ever get her home, that she might pour it all out to her
mother! What a thrilling, wonderful, unheard-of afternoon!
 CHAPTER VI
THE VISION AND THE DREAM

“For a dream cometh through the multitude of business.”

—Ecclesiastes.

As the carriage turned the corner into Third Street, Shackleton and
Mrs. Willers, bidding their adieux to Lepine, started toward The
Trumpet office. The building was not ten minutes’ walk away, and
both the proprietor and the woman reporter had work there that
called them.
In their different ways each was exceedingly elated. The man, with
his hard, bearded face, the upper half shaded by the brim of his soft
felt hat, gave no evidence in appearance or manner of the exultation
that possessed him. But the woman, with her more febrile and less
self-contained nature, showed her excited gratification in her
reddened cheeks and the sparkling animation of her tired eyes. Her
state of joyous triumph was witnessed even in her walk, in the way
she swished her skirts over the pavements, in the something
youthful and buoyant that had crept into the tones of her voice.
“Well,” she said, “that was an experience worth having! I never
heard her sing so before. She just outdid herself.”
“She certainly seemed to me to sing well. I was doubtful at the
beginning, not knowing any more about singing than I do about
Sanskrit, as to whether she really had as fine a voice as we thought.
But there don’t seem to me to be any doubt about it now.”
“Lepine is quite certain, is he?” queried Mrs. Willers, who had tried
to listen to the conversation between her chief and the impresario
on the way out, but had been foiled by Mariposa’s excited chatter.
“He says that she has an unusually fine voice, which, with proper
training, would, as far as they can say now, be perfectly suitable for
grand opera. It’s what they call a dramatic mezzo-soprano, with
something particularly good about the lower notes. Lepine is to see
me again before he goes.”
“Did he suggest what she ought to do?”
“Yes; he spoke of Paris as the best place to send her. He knows
some famous teacher there that he says is the proper person for her
to study with. He seemed to think that two years of study would be
sufficient for her. She’d be ready to make her appearance in grand
opera after that time.”
“Good heavens!” breathed Mrs. Willers in a transport of pious
triumph, “just think of it! And now up in that cottage on Pine Street
getting fifty cents a lesson, and with only four pupils.”
“In two years,” said Shackleton, who was speaking more to himself
than to her, “she’ll be twenty-seven years old—just in her prime.”
“She’ll be twenty-six,” corrected Mrs. Willers; “she’s only twenty-four
now.”
He raised his brows with a little air of amused apology.
“Twenty-four, is it?” he said. “Well, that’s all the better. Twenty-six is
one year better than twenty-seven.”
“It’ll be like the ‘Innocents Abroad’ to see her and her mother in
Paris,” said Mrs. Willers. “They’re just two of the most
unsophisticated females that ever strayed out of the golden age.”
The man vouchsafed no answer to this remark for a moment; then
he said:
“The mother’s health is very delicate? She’s quite an invalid, you
say?”
“Quite. But she’s one of the sweetest, most uncomplaining women
you ever laid eyes on. You’d understand the daughter better if you
knew the mother. She’s so gentle and girlish. And then they’ve lived
round in such a sort of quiet, secluded way. It’s funny to me because
they had plenty of money when Mr. Moreau was alive. But they
never seemed to go into society, or know many people; they just
seemed enough for each other, especially when the father was with
them. They simply adored him, and he must have been a fine man.
They—”
“Is Mrs. Moreau’s state of health too bad to allow her to travel?” said
Shackleton, interrupting suddenly and rudely.
Mrs. Willers colored slightly. She knew her chief well enough to
realize that his tone indicated annoyance. Why did he so dislike to
hear anything about the late Dan Moreau?
“As to that I don’t know,” she said. “She’s so much of an invalid that
she rarely goes out. But with good care she might be able to take a
journey and benefit by it. A sea trip sometimes cures people.”
“Miss Moreau couldn’t, and, I have no doubt, wouldn’t leave her. It’ll
therefore be necessary for the mother to go to Paris with the girl,
and if she is so complete and helpless an invalid she’ll certainly be of
no assistance to her daughter—only a care.”
“She’d undoubtedly be a care. But a person couldn’t separate those
two. They’re wrapped up in each other. It’s a pity you don’t know
Mrs. Moreau, Mr. Shackleton.”
For the second time that afternoon Mrs. Willers was conscious that
words she had intended to be gently ingratiating had given
mysterious offense to her employer. Now he said, with more than an
edge of sharpness to his words:
“I’ve no doubt it’s a pity, Mrs. Willers. But there are so many things
and people it’s a pity I don’t know, that if I came to think it over I’d
probably fall into a state of melancholia. Also, let me assure you,
that I haven’t the least intention of trying to separate Mrs. Moreau
and her daughter. What I’m just now bothered about is the fact that
this lady is hardly of sufficient worldly experience, and certainly has
not sufficient strength to take care of the girl in a strange country.”
“Well, no,” said Mrs. Willers with slow reluctance, “it would be the
other way round, the girl would be taking care of her.”
“That’s exactly what I thought. The only way out of it will be to send
some one with them. A woman who could take care of them both,
chaperone the daughter and look after the mother.”
There was a silence. Mrs. Willers began to understand why Mr.
Shackleton had walked down to The Trumpet office with her. The
walk was over, for they were at the office door, and the conversation
had reached the point to which he had evidently intended to bring it
before they parted.
As they turned into the arched doorway and began the ascent of the
stairs, Mrs. Willers replied:
“I think that would be a very good idea, Mr. Shackleton. That is, if
you can find the right woman.”
“Oh, I’ve got her now,” he answered, giving her a quick, side-long
glance. “I think it would be a good arrangement for all parties. The
Trumpet wants a Paris correspondent.”
The door leading into the press-rooms opened off the landing they
had reached, and he turned into this with a word of farewell, and a
hand lifted to his hat brim. Mrs. Willers continued the ascent alone.
As she mounted upward she said to herself:
“The best thing for me to do is to get a French phrase book on the
way home this evening, and begin studying: ‘Have you the green
pantaloons of the miller’s mother?’”
The elation of his mood was still with Shackleton when, two hours
later, he alighted from the carriage at the steps of his country house.
He went upstairs to his own rooms with a buoyant tread. In his
library, with the windows thrown open to the soft, scented air, he sat
smoking and thinking. The October dusk was closing in, when he
heard the wheels of a carriage on the drive and the sound of voices.
His women-folk with the second of the Thurston girls—the one guest
the house now contained—were returning from the afternoon round
of visits that was the main diversion of their life during the summer
months, and swept the country houses from Redwood City to Menlo
Park.
It was a small dinner table that evening. Winslow had stayed in town
over night, and Shackleton sat at the head of a shrunken board, with
Bessie opposite him, his daughter to the left, and Pussy Thurston on
his right. Pussy was Maud’s best friend and was one of the beauties
of San Francisco. To-night she looked especially pretty in a pale
green crape dress, with green leaves in her fair hair. Her skin was of
a shell-like purity of pink and white, her face was small, with regular
features and a sweet, childish smile.
She and her sister were the only children of the famous Judge
Beauregard Thurston, in his day one of those brilliant lawyers who
brought glory to the California bar. He had made a fortune, lived on
it recklessly and magnificently, and died leaving his daughters almost
penniless. He had been in the heyday of his splendor when Jake
Shackleton, just struggling into the public eye, had come to San
Francisco, and the proud Southerner had not scrupled to treat the
raw mining man with careless scorn. Shackleton evened the score
before Thurston’s death, and he still soothed his wounded pride with
the thought that the two daughters of the man who had once
despised him were largely dependent on his wife’s charity. Bessie
took them to balls and parties, dressed them, almost fed them. The
very green crape gown in which Pussy looked so pretty to-night had
been included in Maud’s bill at a fashionable dressmaker’s.
Personally he liked Pussy, whose beauty and winning manners lent a
luster to his house. Once or twice to-night she caught him looking at
her with a cold, debating glance in which there was little of the
admiration she was accustomed to receiving since the days of her
first long dress.
He was in truth regarding her critically for the first time, for the
Bonanza King was a man on whom the beauty of women cast no
spell. He was comparing her with another and a more regally
handsome girl. Pussy Thurston would look insipid and insignificant
before the stately splendor of his own daughter.
He smiled as he realized Mariposa’s superiority. The young girl saw
the smile, and said with the privileged coquetry of a maid who all
her life has known herself favored above her fellows:
“Why are you smiling all to yourself, Mr. Shackleton? Can’t we know
if it is something pleasant?”
“I was looking at something pretty,” he answered, his eyes full of
amusement as they rested on her charming face. “That generally
makes people smile.”
She was so used to such remarks that her rose-leaf color did not
vary the fraction of a shade. Maud, to whom no one ever paid
compliments, looked at her with wistful admiration.
“Is that all?” she said with an air of disappointment. “I hoped it was
something that would make us all smile.”
“Well, I have an idea that may make you all smile”—he turned to his
wife—“how would you like to go to Europe next spring, Bessie?”
Mrs. Shackleton looked surprised and not greatly elated. On their
last trip to Europe, two years before, her husband had been so
bored by the joys of foreign travel that she had made up her mind
she would never ask him to go again. Now she said:
“But you don’t want to go to Europe. You said last time you hated
it.”
“Did I? Yes, I guess I did. Well, I’m prepared to like it this time. We
could take a spin over in the spring to London and Paris. We’d make
quite a stay in Paris, and you women could buy clothes. You’d come,
too, Pussy, wouldn’t you?” he said, turning to the girl.
Her color rose now and her eyes sparkled. She had never been even
to New York.
“Wouldn’t I?” she said. “That does make me smile.”
“I thought so,” he answered good-humoredly—“and Maud, you’d like
it, of course?”
Maud did not like the thought of going at all. In this little party of
four, two were moved in their actions by secret predilections of
which the others were ignorant. Maud thought of leaving her love
affair at the critical point it had reached, and, with anguish at her
heart, looked heavily indifferent.
“I don’t know,” she said, crumbling her bread, “I don’t think it’s such
fun in Europe. You just travel round in little stuffy trains, and have to
live in hotels without baths.”
“Well, you and I, Pussy,” said Shackleton, “seem to be the only two
who’ve got any enthusiasm. You’ll have to try and put some into
Maud, and if the worst comes to the worst we can kidnap the old
lady.”
He was in an unusually good temper, and the dinner was animated
and merry. Only Maud, after the European suggestion, grew more
stolidly quiet than ever. But she cheered herself by the thought that
the spring was six months off yet, and who could tell what might
happen in six months?
After dinner the ladies repaired to the music room, and Shackleton,
following a custom of his, passed through one of the long windows
into the garden, there to pace up and down while he smoked his
cigar.
The night was warm and odorous with the scent of hidden blossoms.
Now and then his foot crunched the gravel of a path, as his walk
took him back and forth over the long stretch of lawn broken by
flower-beds and narrow walks. The great bulk of the house, its black
mass illumined by congeries of lit windows, showed an inky, irregular
outline against the star-strewn sky.
Presently the sound of a piano floated out from the music room. The
man stopped his pacing, listened for an instant, and then passed
round to the side of the house. The French windows of the music
room were opened, throwing elongated squares of light over the
balcony and the grass beyond. He paused in the darkness and
looked through one of them. There, like a painting framed by the
window casing, was Pussy Thurston seated at the piano singing,
while Maud sat near by listening. One of Miss Thurston’s most
admired social graces was the gift of song. She had a small
agreeable voice, and had been well taught; but the light, frail tones
sounded thin in the wide silence of the night. It was the feebly
pretty performance of the “accomplished young lady.”
Shackleton listened with a slight smile that increased as the song
drew to a close. As it ceased he moved away, the red light of his
cigar coming and going in the darkness.
“Singing!” he said to himself, “they call that singing! Wait till they
hear my daughter!”
 CHAPTER VII
THE REVELATION

“Praised be the fathomless universe

For life and for joy and for objects and knowledge curious,
And for love, sweet love—but praise, praise, praise,
For the sure-enwinding arms of cool-enfolding Death,
The night in silence under many a star,
The ocean shore and the husky whispering wave whose voice I
hear,
And the soul turning to thee, O vast and well-veiled Death,
And the body gratefully nestling close to thee.”

—Whitman.

From the day when Mrs. Willers had appeared with the news of
Shackleton’s interest in her daughter, Lucy’s health had steadily
waned. The process of decay was so quiet, albeit so sure and swift,
that Mariposa, accustomed to the ups and downs of her mother’s
invalid condition, was unaware that the elder woman’s sands were
almost run. The pale intensity, the coldness of the hand gripped
round hers, that had greeted her account of the recital at the Opera-
House, seemed to the girl only the reflection of her own eager
exultation. She was blind, not only from ignorance, but from the
egotistic preoccupations of her youth. It seemed impossible to think
of her mother’s failing in her loving response, now that the sun was
rising on their dark horizon.
But Lucy knew that she was dying. Her feeble body had received its
coup de grâce on the day that Mrs. Willers brought the news of
Shackleton’s wish to see his child. Since then she had spent long
hours in thought. When her mind was clear enough she had
pondered on the situation trying to see what was best to do for
Mariposa’s welfare. The problem that faced her terrified her. The
dying woman was having the last struggle with herself.
One week after the recital at the Opera-House she had grown so
much worse that Mariposa had called in the doctor they had had in
attendance, off and on, since their arrival. He was grave and there
was a consultation. When she saw their faces the cold dread that
had been slowly growing in the girl’s heart seemed suddenly to
expand and chill her whole being. Mrs. Moreau was undoubtedly
very ill, though there was still hope. Yet their looks were sober and
pitying as they listened to the daughter’s reiterated asseverations
that her mother had often been worse and made a successful rally.
An atmosphere of illness settled down like a fog on the little cottage.
A nurse appeared; the doctors seemed to be in the house many
times a day. Mrs. Willers, as soon as she heard, came up, no longer
over-dressed and foolish, but grave and helpful. After a half-hour
spent at Lucy’s bedside, wherein the sick woman had spoken little,
and then only about her daughter, Mrs. Willers had gone to the
office of The Trumpet, frowning in her sympathetic pain. It was
Saturday, and Shackleton had already left for Menlo Park when she
reached the office. But she determined to see him early on Monday
and tell him of the straits of his old friend’s widow and child. Mrs.
Willers knew the signs of the scarcity of money, and knew also the
overwhelming expenses of sickness. What she did not know was
that on Friday morning Mariposa had wept over her check-book, and
then gone out and sold the diamond brooch.
The long Sunday—the interminable day of strained anxiety—passed,
shrouded in rain. When her mother fell into the light sleep that now
marked her condition, Mariposa mechanically went to the window of
the bedroom and looked out. It was one of those blinding rains that
usher in the San Francisco winter, the water falling in straight lances
that show against the light like thin tubes of glass, and strike the
pavement with a vicious impact, which splinters them into spray. It
drummed on the tin roof above the bedroom with an incessant
hollow sound, and ran in a torn ribbon of water from the gutter on
the eaves.
The prospect that the window commanded seemed in dreariness to
match the girl’s thoughts. That part of Pine Street was still in the
unfinished condition described by the words “far out.” Vacant lots
yawned between the houses; the badly paved roadbed was an
expanse of deeply rutted mud, with yellow ponds of rain at the
sewer mouths. The broken wooden sidewalk gleamed with moisture
and was evenly striped with lines of vivid green where the grass
sprouted between the boards. Now and then a wayfarer hurried by,
crouched under the dome of an umbrella spouting water from every
rib.
The gray twilight settled early, and Mariposa, dropping the curtain,
turned to the room behind her. The light of a small fire and a shaded
lamp sent a softened glow over the apartment, which, despite its
poverty, bespoke the taste of gentlewomen in the simple prettiness
of its furnishings. The nurse, a middle-aged woman of a kindly and
capable aspect, sat by the fire in a wicker rocking-chair, reading a
paper. Beside her, on a table, stood the sick-room paraphernalia of
glasses and bottles. The regular creak of the rocking-chair, and an
occasional snap from the fire, were the only sounds that punctuated
the steady drumming of the rain on the tin roof.
A Japanese screen was half-way about the bed, shutting it from the
drafts of the door, and in its shelter Lucy lay sleeping her light,
breathless sleep. In this shaded light, in the relaxed attitude of
unconsciousness, she presented the appearance of a young girl
hardly older than her daughter. Yet the hand of death was plainly on
her, as even Mariposa could now see.
Without sound the girl passed from the room to her own beyond.
Her grief had seized her, and the truth, fought against with the
desperate inexperience of youth, forced itself on her. She threw
herself on her bed and lay there battling with the sickness of despair
that such knowledge brings. Twilight faded and darkness came. In
answer to the servant’s tap on the door, and announcement of
dinner, she called back that she desired none. The room was as dark
about her as her own thoughts. From the door that led into the sick
chamber, only partly closed, a shaft of light cut the blackness, and
on this light she fastened her eyes, swollen with tears, feeling
herself stupefied with sorrow.
As she lay thus on the bed, she heard the creaking of the wicker-
chair as the nurse arose, then came the clink of the spoon and the
glass, and the woman’s low voice, and then her mother’s, stronger
and clearer than it had been for some days. There was an
interchange of remarks between nurse and patient, the sound of
careful steps, and the crack of light suddenly expanded as the door
was opened. Against this background, clear and smoothly yellow as
gold leaf, the nurse’s figure was revealed in sharp silhouette.
“Are you there, Miss Moreau?” she said in a low voice. Mariposa
started with a hurried reply.
“Well, your mother wants to see you and you’d better come. Her
mind seems much clearer and it may not be so again.”
The girl rose from the bed trying to compose her face. In the light of
the open door the woman saw its distress and looked at her
pityingly.
“Don’t tire her,” she said, “but I advise you to say all you have to say.
She may not be this way again.”
Mariposa crossed the room to the bed. Her mother was lying on her
side, pinched, pale and with darkly circled eyes.
“Have you just waked up, darling?” said the girl, tenderly.
“No,” she answered, with a curious lack of response in manner and
tone; “I have been awake some time. I was thinking.”
“Why didn’t you send Mrs. Brown for me? I was in my room passing
the time till you woke up.”
“I was thinking and I wanted to finish. I have been thinking a long
time, days and weeks.”
Mariposa thought her mind was wandering, and sitting down on a
chair by the bedside, took her hand and pressed it gently without
speaking. Her mother lay in the same attitude, her profile toward
her, her eyes looking vacantly at the screen. Suddenly she said:
“You know my old desk, the little rose-wood one Dan gave me? Take
my keys and open it, and in the bottom you’ll see two envelopes,
with no writing. One looks dirty and old. Bring them to me here.”
Mariposa rose wondering, and looking anxiously at her mother. The
elder woman saw the look, and said weakly and almost peevishly:
“Go; be quick. I am not strong enough to talk long. The keys are in
the work-box.”
The girl obeyed as quickly as possible. The desk was a small one
resting on the center-table. It had been a present of her father’s to
her mother, and she remembered it from her earliest childhood in a
prominent position in her mother’s room. She opened it, and in a
few moments, under old letters, memoranda and souvenirs, found
the two envelopes. Carrying them to the bed she gave them to her
mother.
Lucy took them with an unsteady hand, and for a moment lay
staring at her daughter and not moving. Then she said:
“Put the pillows under my head. It’s easier to breathe when I’m
higher,” and as Mariposa arranged them, she added, in a lower
voice: “And tell Mrs. Brown to go; I want to be alone with you.”
Mariposa looked out beyond the screen, and seeing the nurse still
reading the paper, told her to go to the kitchen and get her dinner.
The woman rose with alacrity, and asking Mariposa to call her if the
invalid showed signs of fatigue, or any change, left the room.
The girl turned back to the bedside and took the chair. Lucy had
taken from the dirty envelope a worn and faded paper, which she
slowly unfolded. As she did so, she looked at her daughter with
sunken eyes and said:
“These are my marriage certificates.”
Mariposa, again thinking that her mind was wandering, tried to
smile, and answered gently:
“Your marriage certificate, dear. You were only married once.”
“I was married twice,” said Lucy, and handed the girl the two papers.
Still supposing her mother slightly delirious, the daughter took the
papers and looked at them. The one her eye first fell on was that of
the original marriage. She read the names without at first realizing
whose they were. Then the significance of the “Lucy Fraser” came
upon her. Her glance leaped to the second paper, and at the first
sweep of her eyes over it she saw it was the marriage certificate of
her father and mother, Daniel Moreau and Lucy Fraser, dated at
Placerville twenty-five years before. She turned back to the other
paper, now more than bewildered. She held it near her face, as
though it were difficult to read, and in the dead silence of the room
it began to rustle with the trembling of her hand. A fear of
something hideous and overwhelming seized her. With pale lips she
read the names, and the date, antedating by five years the other
certificate.
“Mother!” she cried, in a wild voice of inquiry, dropping the paper on
the bed.
Lucy, raised on her pillows, was looking at her with a haggard
intentness. All the vitality left in her expiring body seemed
concentrated in her eyes.
“I was married twice,” she said slowly.
“But how? When? What does it mean? Mother, what does it mean?”
“I was married twice,” she repeated. “In St. Louis to Jake
Shackleton, and in Placerville, five years after, to Dan Moreau. And I
was never divorced from Jake. It was not according to the law. I was
never Dan’s lawful wife.”
The girl sat staring, the meaning of the words slowly penetrating her
brain. She was too stunned to speak. Her face was as white as her
mother’s. For a tragic moment these two white faces looked at each
other. The mother’s, with death waiting to claim her, was void of all
stress or emotion. The daughter’s, waking to life, was rigid with
horrified amaze.
Propped by her pillows, Lucy spoke again; her sentences were short
and with pauses between:
“Jake Shackleton married me in St. Louis when I was fifteen. He was
soon tired of me. We went to Salt Lake City. He became a Mormon
there, and took a second wife. She was a waitress in a hotel. She’s
his wife now. He brought us both to California twenty-five years ago.
On the way across, on the plains of Utah, you were born. He is your
father, Mariposa.”
She made an effort and sat up. Her breathing was becoming
difficult, but her purpose gave her strength. This was the
information that for weeks she had been nerving herself to impart.
“He is your father,” she repeated. “That’s what I wanted to tell you.”
Mariposa made no answer, and again she repeated:
“He is your father. Do you understand? Answer me.”
“Yes—I don’t know. Oh, mother, it’s so strange and horrible. And you
sitting there and looking at me like that, and telling it to me! Oh,—
mother!”
She put her hands over her face for an instant, and then dropping
them, leaned over on the bed and grasped her mother’s wrists.
“You’re wandering in your mind. It’s just some hideous dream you’ve
had in your fever. Dearest, tell me it’s not true. It can’t be true. Why,
think of you and me and father always together and with no dreadful
secret behind us like that. Oh—it can’t be true!”
Lucy looked at the papers lying brown and torn on the white quilt.
Mariposa’s eyes followed the same direction, and with a groan her
head sank on her arms extended along the bed. Her mother’s hand,
cold and light, was laid on one of hers, but the dying woman’s face
was held in its quiet, unstirred apathy, as she spoke again:
“Jake was hard to me on the trip. He was a hard man and he never
loved me. After Bessie came he got to dislike me. I was always a
drag, he said. I couldn’t seem to get well after you were born.
Coming over the Sierras we stopped at a cabin. Dan was there with
another man, a miner, called Fletcher. That was the first time I ever
saw Dan.”
Mariposa lifted her head and her eyes fastened on her mother’s face.
The indifference that had held it seemed breaking. A faint smile was
on her lips, a light of reminiscence lit its gray pallor.
“He was always good to anything that was sick or weak. He was
sorry for me. He tried to make Jake stop longer, so I could get
rested. But Jake wouldn’t. He said I had to go on. I couldn’t, but
knew I must, if he said it. We were going to start when Jake said
he’d exchange me for the pair of horses the two miners had in the
shed. So he left me and took the horses.”
“Exchanged you for the horses? Left you there sick and alone?”
“Yes, Jake and Bessie went on with the horses. I stayed. I was too
sick to care.”
She made a slight pause, either from weakness, or in an effort to
arrange the next part of her story.
“I lived there with them for a month. I was sick and they took care
of me. Then one day Fletcher stole all the money and the only horse
and never came back. We were alone there then, Dan and I. I got
better. I came to love him more each day. We were snowed in all
winter, and we lived as man and wife. In the spring we rode into
Hangtown and were married.”
She stopped, a look of ineffable sweetness passed over her face,
and she said in a low voice, as if speaking to herself:
“Oh, that beautiful winter! There is a God, to be so good to women
who have suffered as I had.”
Mariposa sat dumbly regarding her. It was like a frightful nightmare.
Everything was strange, the sick-room, the bed with the screen
around it, her mother’s face with its hollow eyes and pinched nose.
Only the two old dirty papers on the white counterpane seemed to
say that this was real.
Lucy’s eyes, which had been looking back into that glorified past of
love and youth, returned to her daughter’s face.
“But Jake is your father,” she said. “That’s what I had to tell you.
He’ll be good to you. That was why he wanted to find you and help
you.”
“Yes,” said Mariposa, dully, “I understand that now; that was why he
wanted to help me.”
“He’ll be good to you,” went on the low, weak voice, interrupted by
quick breaths. “I know Jake. He’ll be proud of you. You’re handsome
and talented, not weak and poor spirited, as I was. You’re his only
legitimate child; the others are not; they were born in California.
They’re Bessie’s children, and I was his only real wife. You’ll let him
take care of you? Oh, Mariposa, my darling, I’ve told you all this that
you might understand and let him take care of you.”
She made a last call on her strength and leaned forward. Her dying
body was re-vivified; all her mother’s agony of love appeared on her
face. In determining to destroy the illusions of her child to secure
her future, she had made the one heroic effort of her life. It was
done, and for a last moment of relief and triumph she was thrillingly
alive.
Mariposa, in a spasm of despair, threw herself forward on the bed.
“Oh, why did you tell me? Why did you tell me?” she cried. “Why
didn’t you let me think it was the way it used to be? Why did you tell
me?”
Lucy laid her hand on the bowed head.
“Because I wanted you to understand and let him be your father.”
“My father! That man! Oh, no, no!”
“You must promise me. Oh, my beloved child, I couldn’t leave you
alone. It seemed as if God had said to me, ‘Die in peace. Her father
will care for her.’ I couldn’t go and leave you this way, without a
friend. Now I can rest in peace. Promise to let him take care of you.
Promise.”
“Oh, mother, don’t ask me. What have you just told me? That he
sold you to a stranger for a pair of horses, left you to die in a cabin
in the mountains! That’s not my father. My father was Dan Moreau. I
can do nothing but hate that other man now.”
“Don’t blame him, dear, the past is over. Forgive him. Forgive me. If
I sinned there were excuses for me. I had suffered too much. I loved
too well.”
Her voice suddenly hesitated and broke. A gray pallor ran over her
face and a look of terror transfixed her eyes. She straightened her
arms out toward her daughter.
“Promise,” she gasped, “promise.”
With a spring Mariposa snatched the drooping body in her arms and
cried into the face, settling into cold rigidity:
“Yes—yes—I promise! All—anything. Oh, mother, darling, look at me.
I promise.”
She gently shook the limp form, but it was nerveless, only the head
oscillated slightly from side to side.
“Mother, look at me,” she cried frantically. “Look at me, not past me.
Come back to me. Speak to me, I promise everything.”
But there was no response. Lucy lay, limp and white-lipped, her
head lolling back from the support of her daughter’s arm. Her
strength was exhausted to the last drop. She was unconscious.
The wild figure of Mariposa at the kitchen door summoned Mrs.
Brown. Lucy was not dead, but dying. A few moments later Mariposa
found herself rushing hatless through the rain for the doctor, and
then again, in what seemed a few more minutes, standing, soaked
and breathless, by her mother’s side. She sat there throughout the
night, holding the limp hand and watching for a glimmer of
consciousness in the half-shut eyes.
It never came. There was no rally from the collapse which followed
the mother’s confession. She had lived till this was done. Then,
having accomplished the great action of her life, she had loosed her
hold and let go. Once, Mrs. Brown being absent, Mariposa had
leaned down on the pillow and passionately reiterated the assurance
that she would give the promise Lucy had asked. There was a slight
quiver of animation in the dying woman’s face and she opened her
eyes as if startled, but made no other sign of having heard or
understood. But Mariposa knew that she had promised.
On the evening of the day after her confession Lucy died, slipping
away quietly as if in sleep. The death of the simple and unknown
lady made no ripple on the surface of the city’s life. Mrs. Willers and
a neighbor or two were Mariposa’s sole visitors, and the only flowers
contributed to Lucy’s coffin were those sent by the newspaper
woman and Barry Essex. The afternoon of the day on which her
mother’s death was announced, Mariposa received a package from
Jake Shackleton. With it came a short note of condolence, and the
offer, kindly and simply worded, of the small sum of money
contained in the package, which, it was hoped, Miss Moreau, for the
sake of the writer’s early acquaintance with her parents and interest
in herself, would accept. The packet contained five hundred dollars
in coin.
Mariposa’s face flamed. The money fell through her fingers and
rolled about on the floor. She would have liked to take it, piece by
piece, and throw it through the window, into the mud of the street.
She felt that her horror of Shackleton augmented with every passing
moment, gripped her deeper with every memory of her mother’s
words, and every moment’s perusal of the calm, dead face in its
surrounding flowers.
But her promise had been given. She picked up the money and put it
away. Her promise had been given. Already she was beginning dimly
to realize that it would bind and cramp her for the rest of her life.
She was too benumbed now fully to grasp its meaning, but she felt
feebly that she would be its slave as long as he or she lived. But she
had given it.
The money lay untouched throughout the next few days, Lucy’s
simple funeral ceremonies being paid for with the proceeds of the
sale of the diamond brooch, which Moreau had given her in the early
days of their happiness.