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 Contents

Preface to the Fifth Edition xvii

1
The Development of Molecular Biotechnology 1
Emergence of Molecular Biotechnology 1
Recombinant DNA Technology 3
Commercialization of Molecular Biotechnology 4
Concerns and Consequences 7
SUM­MA­RY 9
REFERENCES 9
RE­VIEW QUES­TIONS 10

2
Fundamental Technologies 11
Molecular Cloning 11
Preparation of DNA for Cloning 11
Insertion of Target DNA into a Plasmid Vector 16
Transformation and Selection of Cloned DNA in a Bacterial Host 20
Cloning Eukaryotic Genes 24
Recombinational Cloning 28
Genomic Libraries 30
Genome Engineering us­ing CRISPR Technology 32
Polymerase Chain Reaction 35
Amplification of DNA by PCR 36
Cloning PCR Products 39
Quantitative PCR 39
Chemical Synthesis of DNA 42
Synthesis of Oligonucleotides 42
Assembling Oligonucleotides into Genes 48
Gene Synthesis by PCR 50
DNA Sequencing Technologies 50
Dideoxynucleotide Procedure 53
Pyrosequencing 55
Sequencing using Reversible Chain Terminators 57
Sequencing by Single Molecule Synthesis 57
vii
viii Contents

Sequencing Whole Genomes 59

Preparation of Genomic DNA Sequencing Libraries 60
High-Throughput Next-Generation Sequencing Strategies 61
Genome Sequence Assembly 63
Sequencing Metagenomes 64
Genomics 64
Transcriptomics 67
Proteomics 72
Metabolomics 85
SUM­MA­RY 87
REFERENCES 89
RE­VIEW QUES­TIONS 91

3
Production of Recombinant Proteins 93
Protein Production in Prokaryotic Hosts 93
Regulation of Transcription 94
Increasing Translation Efficiency 98
Increasing Protein Stability 102
Increasing Protein Secretion 106
Facilitating Protein Purification 110
DNA Integration into the Host Chromosome 115
Heterologous Protein Production in Eukaryotic Cells 120
Posttranslational Modification of Eukaryotic Proteins 120
Ge­ne­ral Features of Eukaryotic Expression Systems 122
Yeast Expression Systems 124
Baculovirus–Insect Cell Expression Systems 136
Mammalian Cell Expression Systems 143
Protein Engineering 153
Directed Mutagenesis 154
Random Mutagenesis 158
Examples of Protein Engineering 162
SUM­MA­RY 171
REFERENCES 173
RE­VIEW QUES­TIONS 175

4
Molecular Diagnostics 177
Immunological Approaches to Detect Protein Biomarkers 178
Antibodies 178
Agglutination 183
Enzyme-Linked Immunosorbent Assays 183
Protein Arrays to Detect Polygenic Diseases 189
Immunoassays for Protein Conformation-Specific Disorders 191
DNA-Based Diagnostic Approaches 193
Hybridization Probes 193
PCR-Based Detection Methods 200
DNA Microarrays 208
Whole Genome Sequencing to Assess Genetic Disease Risk 214
 Contents ix

Detecting RNA Signatures of Disease 215

Detection of Disease-Associated Changes in Gene Expression
Using Microarrays 215
Detection of RNA Signatures of Antibiotic Resistance in Bacteria 216
Detection of miRNA Signatures of Cancers 217
Biofluorescent and Bioluminescent Systems 219
Fluorescent Pro­teins 219
Luciferase 221
Microbial Biosensors 222
SUM­MA­RY 224
REFERENCES 225
RE­VIEW QUES­TIONS 227

5
Protein Therapeutics 229
Pharmaceuticals 230
Human Interferons 231
Human Growth Hormone 235
Tumor Necrosis Factor Alpha 237
Extending Protein Half-Life 238
Enzymes 240
DN­ase I 240
Alginate Lyase 242
Phenylalanine Ammonia Lyase 245
α1-Antitrypsin 247
Glycosidases 248
Masking Nonhuman Epitopes 249
Engineering Bacteriophages 250
Targeting Mitochondria 253
Lactic Acid Bacteria 255
Interleukin-10 255
Leptin 258
An HIV Inhibitor 258
Insulin 260
Recombinant Antibodies 261
Hybrid Human–Mouse Monoclonal Antibodies 264
Human Monoclonal Antibodies 268
Antibody Fragments 270
Combinatorial Libraries of Antibody Fragments 274
A Combinatorial Library of Full-Length Antibodies 277
Shuffling CDR Sequences 278
Dual-Variable-Domain Antibodies 280
Anticancer Antibodies 281
Antibodies Against Various Diseases 284
Antiobesity Antibodies 287
Enhanced Antibody Half-Life 290
SUM­MA­RY 292
REFERENCES 292
RE­VIEW QUES­TIONS 295
x Contents

6
Nucleic Acids as Therapeutic Agents 297
Targeting Specific mRNA and DNA Se­quences 299
Antisense RNA 299
Aptamers 302
Ribozymes and DNAzymes 307
Interfering RNA 311
Zinc Finger Nucleases 315
CRISPR-Cas System 317
Nanozymes 318
Nanoparticles 319
Viral Delivery Systems 319
Nonviral Delivery Systems 325
Direct Injection 325
Lipids 327
Bacteria 328
Dendrimers 331
Antibodies 332
Aptamers 332
Transposons 334
Gene Therapy 335
Prodrug Activation Therapy 335
Promoterless Gene Targeting 337
SUM­MA­RY 338
REFERENCES 339
RE­VIEW QUES­TIONS 341

7
Vaccines 343
Vaccination 343
Current and Future Vaccines 345
Subunit Vaccines 347
Herpes Simplex Virus 348
Cholera 350
SARS 350
Staphylococcus au­re­us 351
Human Papillomavirus 353
Foot-and-Mouth Virus 354
Streptococcus 356
Delivery 357
Peptide Vaccines 359
Malaria 359
Genetic Immunization: DNA Vaccines 363
Delivery 364
Cancer 370
Dental Caries 370
Attenuated Vaccines 372
Herpes Simplex Virus 372
Cholera 374
 Contents xi

Salmonella Species 375

Leishmania Species 378
Vector Vaccines 378
Vaccines Directed against Viruses 378
Vaccines Directed against Bacteria 388
Bacteria as Antigen Delivery Systems 392
Monoclonal Antibody Passive Immunity 396
Influenza Virus 396
SUM­MA­RY 397
REFERENCES 398
RE­VIEW QUES­TIONS 400

8
Industrial and Environmental Uses of
Recombinant Microorganisms 403
Restriction Endonucleases 403
Small Biological Molecules 405
L-Ascorbic Acid 407
Indigo 410
Amino Acids 412
Lycopene 417
Antibiotics 418
Biopolymers 429
Microbial Degradation of Xenobiotics 434
Genetic Engineering of Biodegradative Pathways 436
Utilization of Starch and Sugars 445
Commercial Production of Fructose and Alcohol 446
Increasing Alcohol Production 448
Improving Fructose Production 453
Utilization of Cellulose and Hemicellulose 454
Lignocellulosics 455
Cellulase Genes 457
Direct Conversion of Biomass to Ethanol 462
Zymomonas mobilis 464
Lipids from Cyanobacteria 467
Hydrogen Production 468
SUM­MA­RY 470
REFERENCES 471
RE­VIEW QUES­TIONS 474

9
Large-Scale Production of Proteins from
Recombinant Microorganisms 475
Principles of Microbial Growth 476
Batch Fermentation 477
Fed-Batch Fermentation 479
Continuous Fermentation 480
xii Contents

Maximizing The Efficiency of The Fermentation Process 481

High-Density Cell Cultures 483
Increasing Plasmid Stability 484
Quiescent E. Coli Cells 485
Protein Secretion 486
Reducing Acetate 489
Bioreactors 491
Typical Large-Scale Fermentation Systems 494
Two-Stage Fermentation in Tandem Airlift Reactors 495
Two-Stage Fermentation in a Single Stirred-Tank Reactor 496
Batch ver­sus Fed-Batch Fermentation 498
Harvesting Microbial Cells 501
Disrupting Microbial Cells 502
Downstream Processing 504
Protein Solubilization 506
Utilizing an Immobilized Enzyme 507
Magnetic Separation of Proteins 507
Large-Scale Production of Plasmid DNA 508
SUM­MA­RY 511
REFERENCES 512
RE­VIEW QUES­TIONS 514

10
Genetic Engineering of Plants: Methodology 515
Plant Transformation with the Ti Plasmid of
A. Tumefaciens 516
Ti Plasmid-Derived Vector Systems 522
Microprojectile Bombardment 526
Chloroplast Engineering 527
Very High Level Protein Expression 529
Use of Reporter Genes in Transformed Plant Cells 532
Manipulation of Gene Expression in Plants 533
Transient Gene Expression 533
Plant Promoters 536
Targeted Gene Editing 538
Facilitating Protein Purification 539
Protein Glycosylation 541
Production of Marker-Free Transgenic Plants 542
Removing Marker Genes from Nuclear DNA 543
Removing Marker Genes from Chloroplast DNA 545
SUM­MA­RY 546
REFERENCES 547
RE­VIEW QUES­TIONS 549
 Contents xiii

11
Transgenic Plants 551
Insect Resistance 551
B. thuringiensis Insecticidal Toxin 551
Increasing Expression of the B. thuringiensis Protoxin 555
Other Strategies for Protecting Plants against Insects 558
Preventing the Development of B. thuringiensis-Resistant
Insects 564
Targeting Aphids 569
Virus Resistance 570
Viral Coat Protein-Mediated Protection 570
Protection by Expression of Other Genes 574
Herbicide Resistance 578
Fungus and Bacterium Resistance 583
Salt and Drought Stress 588
Fruit Ripening and Flower Wilting 592
Modification of Plant Nutritional Content 594
Amino Acids 594
Lipids 595
Vitamins 599
Iron 601
Gluten 602
Modification of Food Plant Taste and Appearance 603
Preventing Discoloration 603
Starch 605
Plants as Bioreactors 608
Antibodies 608
Poly(3-hydroxybutyric Acid) 610
Eddible Vaccines 611
Plant Yield 615
Altering Lignin Content 615
Increasing Oxygen Content 618
SUM­MA­RY 619
REFERENCES 620
RE­VIEW QUES­TIONS 624

12
Transgenic Animals 625
Transgenic Mice: Methodology 626
DNA Microinjection Method 627
Retroviral Vector Method 629
Engineered Embryonic Stem Cell Method 631
Conditional Gene Inactivation with the Cre–loxP
Recombination System 637
Genome Editing with the CRISPR-Cas Sys­tem 641
Gene Knockdown by RNA Interference 643
xiv Contents

Transgenic Mice: Applications 644

Transgenic Disease Models: Alzheimer Disease 644
Transgenic Mice as Test Systems 647
Control of Transgene Expression 651
Conditional Control of Cell Death 654
Transgenic Livestock 656
Cloning Livestock by Somatic Cell Nuclear Transfer 656
Production of Pharmaceuticals 658
Production of Donor Organs 660
Disease Resistant Livestock 661
Improving Milk Quality 664
Improving Animal Production Traits 665
Transgenic Poultry 669
Transgenic Fish 673
SUM­MA­RY 676
REFERENCES 676
RE­VIEW QUES­TIONS 678

13
Molecular Biotechnology and Society 679
Development of Guidelines for Recombinant
DNA Research 680
Deliberate Release of Genetically Modified
Microorganisms 682
Environmental Concerns 682
Regulations 683
Regulation of Genetically Modified Foods 684
Food Ingredients Produced by Genetically Engineered
Microorganisms 684
Genetically Modified Crops 687
Genetically Engineered Livestock 691
Societal Concerns About Genetically Modified Foods 692
Alteration of Nutritional Content of Food 692
Potential for Introducing Toxins or Allergens into Food 696
Potential for Transferring Transgenes from Food to Humans
or Intestinal Microorganisms 698
Controversy About the Labeling of Genetically Modified Foods 700
Impact of Genetically Engineered Crops on Biodiversity 700
Who Benefits from Production of Genetically Modified Foods? 703
Environmental Benefits of Genetically Modified Crops 704
How do Views about Genetically Engineered Organisms
Impact Trade? 705
Regulation and Safety of Medical Products of
Biotechnology 706
New Biological Drugs 706
Genetic and Genomic Testing 709
Economic Issues 711
 Contents xv

Patenting Biotechnology 714

Patenting 714
Patenting in Different Countries 716
Patenting Nucleic Acid Sequences 717
Patenting Living Organisms 719
Patenting and Fundamental Research 720
SUM­MA­RY 721
REFERENCES 722
RE­VIEW QUES­TIONS 724

Amino Acids of Proteins and Their Designations 725

Index 727
Preface to the Fifth Edition

Based on the de­vel­op­ment of re­com­bi­nant DNA tech­nol­ogy, mo­lec­u­lar

bio­tech­nol­ogy emerged as a new re­search dis­ci­pline in the late 1970’s.
Since those early days, there has been a ver­i­ta­ble ex­plo­sion of knowl­edge
in the bi­o­log­i­cal sci­ences. With the advent of PCR, chem­i­cal DNA syn­the­sis,
DNA se­quenc­ing, mono­clo­nal an­ti­bod­ies, di­rected mu­ta­gen­e­sis, ge­no­mics,
pro­te­o­mics, metabolomics, and more re­cently, spe­cific ge­nome mod­i­fi­ca­t ion
tech­niques, our un­der­stand­ing of and abil­ity to ma­nip­u­late the bi­o­log­i­cal
world has grown ex­po­nen­tially. When the first edi­tion of Molecular Biotech-
nology: Principles and Applications of Recombinant DNA was pub­lished in 1994,
nearly all­of the trans­genic or­gan­isms that were pro­duced in­cluded only a
sin­gle in­tro­duced gene. Now, 23 years later it is com­mon for re­search­ers to
en­gi­neer or­gan­isms by both mod­i­fy­ing the ac­tiv­ity and the reg­u­la­tion of
ex­ist­ing genes and also by in­tro­duc­ing en­tire new path­ways. In 1994, only a
hand­ful of prod­ucts pro­duced by this new tech­nol­ogy had been com­mer­
cial­ized. Today, as a con­se­quence of mo­lec­u­lar bio­tech­nol­ogy hun­dreds of
new ther­a­peu­tic agents are avail­­able in the mar­ket­place with many more in
the pipe­line as well as doz­ens of trans­genic plants. DNA tech­nol­o­gies have
be­come a cor­ner­stone of mod­ern fo­ren­sics, pa­ter­nity test­ing and an­ces­try
de­ter­m i­na­t ion. A num­ber of new re­com­bi­nant vac­cines have been de­vel­
oped, with many more on the ho­ri­zon. The list goes on and on. Molecular
bio­tech­nol­ogy has clearly lived up to its prom­ise and all­of the orig­i­nal hype
that has ex­isted since the late 1970s. Worldwide there are sev­eral thou­sand
bio­tech­nol­ogy com­pa­nies, in vir­tu­ally ev­ery cor­ner of the globe, em­ploy­ing
hun­dreds of thou­sands of sci­en­tists. When the ex­cit­ing sci­ence be­ing done
at uni­ver­si­ties, gov­ern­ment labs and re­search in­sti­tutes around the world is
fac­tored in, the rate of change and of dis­cov­ery in the bi­o­log­i­cal sci­ences is
ab­so­lutely as­tound­ing. This fifth edi­t ion of Molecular Biotechnology, build­
ing upon the fun­da­men­tals that were es­tab­lished in the pre­vi­ous four
edi­tions, en­deav­ors to pro­vide read­ers with a win­dow on some of the ma­
jor de­vel­op­ments in this grow­ing field. Given the enor­mity of the field of
mo­lec­u­lar bio­tech­nol­ogy, we have had to be highly se­lec­tive in the ma­te­rial
we in­cluded in this edi­t ion. Moreover, the win­dow that we are look­ing
through is mov­ing. This not­with­stand­ing, we both ex­pect and look for­ward
to the com­mer­cial­i­za­tion of many of the dis­cov­er­ies that are dis­cussed here,
and in the fu­ture to the de­vel­op­ment of many new ap­proaches, in­sights,
and dis­cov­er­ies.

xvii
xviii Preface to the Fifth Edition

We have through­out­en­deav­ored to make the text reader friendly by min­

i­miz­ing the use of tech­ni­cal jar­gon and un­nec­es­sary ab­bre­vi­a­tions. Moreover,
when an im­por­tant term ap­pears for the first time in the text, it is fol­lowed in
pa­ren­the­ses with a syn­o­nym or brief ex­pla­na­tion. The over­all size of this edi­
tion has been pared down significantly com­pared to the fourth edi­tion, done,
in large mea­sure, by re­mov­ing some older ma­te­rial that has come to be com­
mon knowl­edge within the past 10–20 years. In ad­di­tion, to fa­cil­i­tate the
book’s flow and ease of un­der­stand­ing, in a num­ber of in­stances, two or more
fig­ures have been com­bined into a sin­gle fig­ure. Endeavoring to be as up-to-
date as pos­si­ble, this edi­tion ex­pands the dis­cus­sion of in­ter­fer­ing RNA and
ex­plains CRISPR tech­nol­ogy in de­tail, pro­vid­i ng ex­am­ples of their use in
both gene ther­apy and trans­genic plants.
Each chap­ter opens with an out­­line of top­ics and con­cludes with a sum­
mary and list of re­view ques­t ions to sharpen stu­dents’ crit­i­cal think­ing
skills. All of the key ideas in the book are il­lus­trated by the more than 500
full-color fig­ures and elab­o­rated in more than 80 tables. After in­tro­duc­ing
mo­lec­u­lar bio­tech­nol­ogy as a sci­en­tific and eco­nomic ven­ture in Chapter 1,
the next two chap­ters ex­plain the de­tailed meth­od­ol­o­g ies of mo­lec­u­lar
bio­tech­nol­ogy. These chap­ters pro­vide a solid sci­en­tific base for the re­
main­der of the book. Chapters 4 to 8 pres­ent ex­am­ples of mi­cro­bial mo­lec­
u­lar bio­tech­nol­ogy cov­er­ing such top­ics as the pro­duc­tion of me­tab­o­lites,
new vac­cines, both pro­tein and nu­cleic acid ther­a­peu­tic agents, di­ag­nos­tics,
bio­re­me­di­a­tion, and bio­mass uti­li­za­tion. Chapter 9 de­scribes some of the
key com­po­nents of large-scale fer­men­ta­t ion pro­cesses us­i ng re­com­bi­nant
mi­cro­or­gan­isms. Chapters 10 to 12 de­scribe the mo­lec­u­lar bio­tech ­nol­ogy
of plants and an­i ­mals. The book con­c ludes in Chapter 13 with a dis­cus­
sion of the in­ter­ac­tion of mo­lec­u­lar bio­tech ­nol­ogy with so­ci­ety in­clud­i ng
con­tro­ver­sies that have oc­curred as a con­se­quence of this tech­nol­ogy, cov­
er­age of the reg­u­la­tion of mo­lec­u­lar bio­tech ­nol­ogy and pat­ents.
Throughout the text we have re­lied ex­ten­sively upon the re­cent pub­
lished work of many re­search­ers. In all­cases, al­though not cited di­rectly in
the body of a chap­ter, the orig­i­nal pub­lished ar­ti­cles are cited in the refer-
ences sec­tion of the ap­pro­pri­ate chap­ter. In some cases, we have taken
“ped­a­gogic li­cense” and ei­t her ex­t racted or re­for­mu­lated data from the
orig­i­nal pub­li­ca­tions. Clearly, we are re­spon­si­ble for any dis­tor­tions or mis­
rep­re­sen­ta­tions from these sim­pli­fi­ca­tions, al­though we hope that none has
oc­curred. The references sec­tions also con­tain other sources that we used
in a gen­eral way, which might, if con­sulted, bring the read­ers closer to a
par­t ic­u­lar sub­ject.
Ber­nard R. Glick
Cheryl L. Patten
 doi: 10.1128/9781555819378.ch1

Emergence of Molecular
Biotechnology
Recombinant DNA Technology
Commercialization of Molecular
Biotechnology
The Development of
Molecular Biotechnology

Emergence of Molecular Biotechnology

1
Long be­fore we knew that mi­cro­or­gan­isms ex­isted or that genes were the
units of in­her­i­tance, hu­mans looked to the nat­u­ral world to de­velop meth­
Concerns and Consequences
SUMMARY ods to in­crease food pro­duc­tion, pre­serve food, and heal the sick. Our an­
REFERENCES ces­tors dis­cov­ered that grains could be pre­served through fer­men­ta­tion
REVIEW QUESTIONS into beer, that stor­ing horse sad­dles in a warm, damp cor­ner of the sta­ble
re­sulted in the growth of a sad­dle mold that could heal in­fected sad­dle
sores, that in­ten­tional ex­po­sure to a “con­ta­gion” could some­how pro­vide
pro­tec­tion from an in­fec­tious dis­ease on sub­se­quent ex­po­sures, and that
plants and an­i­mals with en­hanced pro­duc­tion traits could be de­vel­oped
through cross breed­ing. Following the dis­cov­ery of the mi­cro­scopic world
in the 17th cen­tury, mi­cro­or­gan­isms have been em­ployed in the de­vel­op­
ment of nu­mer­ous use­ful pro­cesses and prod­ucts. Many of these are found
in our house­holds and back­yards. Lactic acid bac­te­ria are used to pre­pare
yo­gurts and pro­bi­ot­ics, in­sec­ti­cide-producing bac­te­ria are sprayed on
many of the plants from which the veg­e­ta­bles in our re­frig­er­a­tor are har­
vested, ni­tro­gen-fixing bac­te­ria are added in the soil used for cul­ti­va­tion of
le­gumes, the en­zy­matic stain re­mov­ers in laun­dry de­ter­gent come from a
mi­cro­or­gan­ism, and an­ti­bi­ot­ics that are de­rived from com­mon soil mi­
crobes are used to treat in­fec­tious dis­eases. These are just a few ex­am­ples
of tra­di­tional bio­tech­nol­o­gies that have im­proved our lives. Up to the early
1970s, how­ever, tra­di­tional bio­tech­nol­ogy was not a well-recognized sci­
en­t ific dis­ci­pline, and re­search in this area was cen­tered in de­part­ments
of chem­i­cal en­g i­neer­i ng and oc­ca­sion­ally in spe­cial­i zed mi­cro­bi­ol­ogy
pro­grams.
In a broad sense, bio­tech­nol­ogy is con­cerned with the ma­nip­u­la­tion of
or­gan­isms to de­velop and man­u­fac­ture use­ful prod­ucts. The term “bio­
tech­nol­o­gy” was first used in 1917 by a Hun­gar­ian en­gi­neer, Karl Ereky, to
de­scribe an in­te­grated pro­cess for the large-scale pro­duc­tion of pigs by

1
2 Chapter 1

us­ing sugar beets as the source of food. According to Ereky, bio­tech­nol­ogy

was “all­lines of work by which prod­ucts are pro­duced from raw ma­te­ri­als
with the aid of liv­ing things.” This fairly pre­cise def­i­ni­tion was more or less
ig­nored. For a num­ber of years, bio­tech­nol­ogy was used to de­scribe two
very dif­fer­ent en­gi­neer­i ng dis­ci­plines. On one hand, it re­ferred to in­dus­
trial fer­men­ta­tion. On the other, it was used for the study of ef­fi­ciency in the
work­place—what is now called er­go­nom­ics. This am­bi­gu­ity ended in 1961
when the Swed­ish mi­cro­bi­ol­o­gist Carl Göran Hedén rec­om­mended that
the ti­tle of a sci­en­tific jour­nal ded­i­cated to pub­lish­ing re­search in the fields
of ap­plied mi­cro­bi­ol­ogy and in­dus­trial fer­men­ta­tion be changed from the
Journal of Microbiological and Biochemical Engineering and Technology to Bio-
technology and Bioengineering. From that time on, bio­tech­nol­ogy has been
de­fined as the ap­pli­ca­tion of sci­en­tific and en­gi­neer­ing prin­ci­ples to the
pro­cess­ing of ma­te­rial by bi­o­log­i­cal agents to pro­vide goods and ser­vices. It
is grounded on ex­per­tise in mi­cro­bi­ol­ogy, ge­net­ics, bio­chem­is­try, im­mu­
nol­ogy, cell bi­­ol­ogy, and chem­i­cal en­gi­neer­ing.
Commodity pro­duc­tion by nat­u­rally oc­cur­ring mi­cro­bial strains on a
large scale is of­ten con­sid­er­ably less than op­ti­mal. Initial ef­forts to en­
hance prod­uct yields fo­cused on cre­at­ing var­i­ants (mu­tants) us­ing chem­i­cal
mu­ta­gens or ra­di­a­tion to in­duce changes in the ge­netic con­sti­tu­tion of ex­ist­
ing strains. However, the level of im­prove­ment that could be achieved in
this way was usu­ally lim­ited bi­o­log­i­cally. If a mu­tated strain, for ex­am­ple,
syn­the­sized too much of a com­pound, other met­a­bolic func­tions of­ten
were im­paired, thereby caus­i ng the strain’s growth dur­i ng large-scale fer­
men­ta­tion to be less than de­sired. Despite this con­straint, the tra­di­tional
“in­duced mu­ta­gen­e­sis and se­lec­tion” strat­e­gies of strain im­prove­ment were
ex­tremely suc­cess­ful for a num­ber of pro­cesses, such as the pro­duc­tion of
an­ti­bi­ot­ics.
The tra­di­t ional ge­netic im­prove­ment reg­i­mens were te­dious, time-
con­sum­ing, and costly be­cause of the large num­bers of mi­cro­bial cells that
had to be screened and tested. Moreover, the best re­sult that could be ex­pected
with this ap­proach was the im­prove­ment of an ex­ist­ing in­her­ited prop­erty of
a mi­cro­or­gan­ism rather than the ex­pan­sion of its ge­netic ca­pa­bil­i­ties. Despite
these lim­i­ta­tions, by the late 1970s ef­fec­tive pro­cesses for the mass pro­duc­tion
of a wide range of com­mer­cial prod­ucts had been per­fect­ed.
Today we have ac­quired suf­fi­cient knowl­edge of the bio­chem­is­try, ge­net­ics,
and mo­lec­u­lar bi­­ol­ogy of mi­crobes and other or­gan­isms to ac­cel­er­ate the
de­vel­op­ment of use­ful and im­proved bi­o­log­i­cal prod­ucts and pro­cesses and
to cre­ate new prod­ucts that would not oth­er­wise oc­cur. Distinct from tra­di­
tional bio­tech­nol­ogy, the mod­ern meth­ods re­quire knowl­edge of and ma­
nip­u­la­tion of genes, the func­tional units of in­her­i­tance, and the dis­ci­pline
that is con­cerned with the ma­nip­u­la­tion of genes for the pur­pose of pro­duc­
ing use­ful goods and ser­vices us­ing liv­ing or­gan­isms is known as mo­lec­u­
lar bio­tech­nol­ogy. The piv­otal de­vel­op­ments that en­abled this tech­nol­ogy
were the es­tab­lish­ment of tech­niques to iso­late genes and to trans­fer them
from one or­gan­ism to an­other. This tech­nol­ogy is known as re­com­bi­nant
DNA tech­nol­ogy, and it be­gan as a lunch­time con­ver­sa­tion be­tween two sci­
en­tists work­ing in dif­fer­ent fields who met at a sci­en­tific con­fer­ence in 1972.
In his lab­o­ra­tory at Stan­ford University in Cal­i­for­nia, Stan­ley Co­hen had
been de­vel­op­i ng meth­ods to trans­fer plas­m ids, small cir­cu­lar DNA mol­
e­cules, into bac­te­r ial cells. Meanwhile, Her­bert Boyer at the University of
Cal­i­for­nia at San Fran­cisco was work­ing with en­zymes that cut DNA at
 The Development of Molecular Biotechnology 3

spe­cific nu­cle­o­tide se­quences. Over lunch at a sci­en­tific meet­ing in Ha­waii,

they rea­soned that Boy­er’s en­zyme could be used to splice a spe­cific seg­
ment of DNA into a plas­mid and then the re­com­bi­nant plas­mid could be
in­tro­duced into a host bac­te­rium us­ing Co­hen’s meth­od.

Recombinant DNA Technology

It was clear to Co­hen and Boyer, and oth­ers, that re­com­bi­nant DNA tech­
nol­ogy had far-reach­ing pos­si­bil­i­ties. As Co­hen noted at the time, “It may
be pos­si­ble to in­tro­duce in E. coli, genes spec­i­f y­i ng met­a­bolic or syn­thetic
func­tions such as pho­to­syn­the­sis or an­ti­bi­otic pro­duc­tion in­dig­e­nous to
other bi­o­log­i­cal clas­ses.” The first com­mer­cial prod­uct pro­duced us­ing re­
com­bi­nant DNA tech­nol­ogy was hu­man in­su­lin, which is used in the
treat­ment of di­a­be­tes. The DNA se­quence that en­codes hu­man in­su­lin
was syn­the­sized, a re­mark­able feat in it­self at the time, and was in­serted
into a plas­mid that could be main­tained in a non­patho­genic strain of the
bac­te­rium E. coli. The bac­te­rial host cells acted as bi­o­log­i­cal fac­to­ries for the
pro­duc­tion of the two pep­tide chains of hu­man in­su­lin that, af­ter com­bin­
ing, could be pu­ri­fied and used to treat di­a­bet­ics who were al­ler­gic to the

Construction of Biologically Functional Bacterial

Plasmids In Vitro
Co­hen SN, Chang ACY, Boyer HW, Helling RB. 1973.
Proc. Natl. Acad. Sci. USA 70:3240–3244.
The land­mark study of Co­h en et al. es­
tab­lished the foun­da­tion for re­com­b i­
nant DNA tech­ n ol­
o gy by show­ ing
how ge­n etic in­for­ma­tion from dif ­fer­
ent sources could be joined to cre­ate a
novel, rep­li­ca­ble ge­netic struc­ture. In
this in­stance, the new ge­netic en­ti­ties
were de­rived from bac­te­rial au­ton­o ­
mously rep­li­cat­ing ex ­tra­chro­mo­somal
DNA struc­ tures called plas­ mids. In a
pre­vi­ous study, Co­hen and Chang (Proc.
Natl. Acad. Sci. USA. 70:1293–1297, 1973)
pro­duced a small plas­mid from a large
nat­u­rally oc­cur­ring plas­mid by shear­ing
the larger plas­mid into smaller ran­dom
pieces and in­tro­duc­ing the mix­ture of
pieces into a host cell, the bac­te­rium E.
coli. By chance, one of the frag­ments that
was about ; the size of the orig­i­nal
plas­mid was per­pet­u­ated as a func­tional
plas­mid. To over­come the ran­dom­ness
of this ap­proach and to make the ge­
netic ma­nip­u­la­tion of plas­mids more
man­age­able, Co­hen and his co­work­ers
de­cided to use an en­zyme (re­stric­tion
en­do­nu­cle­ase) that cuts a DNA mol­e­cule
at a spe­cific site and pro­duces a short ex­
ten­sion at each end. The ex­ten­sions of
the cut ends of a re­stric­tion en­do­nu­cle­
ase-treated DNA mol­e­cule can com­bine
with the ex­ten­sions of an­other DNA mol­
e­cule that has been cleaved with the
same re­stric­tion en­do­nu­cle­ase. Conse­
quently, when DNA mol­e­cules from dif­
fer­ent sources are treated with the same
re­stric­tion en­do­nu­cle­ase and mixed to­
gether, new DNA com­ bi­
na­
tions that
never ex­isted be­fore can be formed. In
this way, Co­hen et al. not only in­tro­duced
a gene from one plas­mid into an­other
plas­mid but also dem­on­strated that the
in­tro­duced gene was bi­o­log­i­cally ac­tive.
To their credit, these au­thors fully ap­pre­
ci­ated that their strat­egy was “po­ten­tially
use­ ful for in­ ser­tion of spe­ cific se­
quences from pro­kary­otic or eu­kary­otic
milestone
chro­mo­somes or ex­tra­chro­mo­somal
DNA into in­de­pen­dently rep­li­cat­ing bac­
te­rial plas­mids.” In other words, any gene
from any or­gan­ism could the­o­ret­i­cally be
cloned into a plas­mid which, af­ter in­tro­
duc­tion into a host cell, would be main­
tained in­def­i­nitely and, per­haps, pro­duce
the pro­tein en­coded by the cloned gene.
By dem­on­strat­ing the fea­si­bil­ity of gene
clon­ing, Co­hen et al. pro­vided the ex­per­i­
men­tal ba­sis for re­com­bi­nant DNA tech­
nol­ogy and es­tab­lished that plas­mids
could act as ve­hi­cles (vec­tors) for main­
tain­ing cloned genes. This mo­ ti­
vated
oth­ers to pur­sue re­search in this area that
rap­idly led to the de­vel­op­ment of more
so­phis­ti­cated vec­tors and gene clon­ing
strat­e­gies. It also en­gen­dered con­cerns
about the safety and eth­ics of this kind of
re­search that, in turn, were re­spon­si­ble
for the es­tab­lish­ment of of ­fi­cial guide­
lines and gov­ern­men­tal agen­cies for con­
duct­ing and reg­u­lat­ing re­com­bi­nant
DNA re­search, re­spec­tive­ly; and con­trib­
uted to the for­ma­tion of the mo­lec­u­lar
bio­tech­nol­ogy in­dus­try.
4 Chapter 1

com­mer­cially avail­­able por­cine (pig) in­su­lin. Today, this type of ge­netic en­
gi­neer­ing is com­mon­place.
The na­ture of bio­tech­nol­ogy was changed for­ever by the de­vel­op­ment of
re­com­bi­nant DNA tech­nol­ogy. Genetic en­gi­neer­ing pro­vided the means to
cre­ate, rather than merely iso­late, highly pro­duc­tive mi­cro­bial strains. Not
long af­ter the pro­duc­tion of the first com­mer­cial prep­a­ra­tion of re­com­bi­
nant hu­man in­su­lin in 1982, bac­te­ria and then eu­kary­otic cells were used
for the pro­duc­tion of in­su­lin, in­ter­feron, growth hor­mone, vi­ral an­ti­gens,
and a va­ri­ety of other ther­a­peu­tic pro­teins. Recombinant DNA tech­nol­ogy
also fa­cil­i­tated the bi­o­log­i­cal pro­duc­tion of large amounts of use­ful low-
molecular-weight com­pounds and mac­ro­mol­e­cules that oc­cur nat­u­rally in
mi­nus­cule quan­ti­ties. Plants and an­i­mals be­came tar­gets as nat­u­ral bio­re­
ac­tors for pro­duc­ing new or al­tered gene prod­ucts that could never have
been cre­ated ei­ther by mu­ta­gen­e­sis and se­lec­tion or by cross­breed­i ng. Mo­
lecular bio­tech ­nol­ogy has be­come the stan­dard method for de­vel­op­i ng
liv­ing sys­tems with novel func­tions and ca­pa­bil­i­ties for the syn­the­sis of
im­por­tant com­mer­cial prod­ucts.
Most new sci­en­tific dis­ci­plines do not arise solely on their own. They are
of­ten formed by the syn­the­sis of knowl­edge from dif­fer­ent ar­eas of re­
search. For mo­lec­u­lar bio­tech­nol­ogy, the bio­tech­nol­ogy com­po­nent was
per­fected by in­dus­trial mi­cro­bi­ol­o­gists and chem­i­cal en­gi­neers, whereas
the re­com­bi­nant DNA tech­nol­ogy por­tion owes much to dis­cov­er­ies in mo­
lec­u­lar bi­­ol­ogy, bac­te­rial ge­net­ics, and nu­cleic acid en­zy­mol­ogy (Table 1.1).
In a broad sense, mo­lec­u­lar bio­tech­nol­ogy draws on knowl­edge from a
di­verse set of fun­da­men­tal sci­en­tific dis­ci­plines to cre­ate prod­ucts that are
use­ful in a wide range of ap­pli­ca­tions (Fig. 1.1).
The Co­hen and Boyer strat­egy for gene clon­ing was an ex­per­i­ment
“heard round the world.” Once their con­cept was made pub­lic, many other
re­search­ers im­me­di­ately ap­pre­ci­ated its po­ten­tial. Consequently, sci­en­tists
cre­ated a large va­ri­ety of ex­per­i­men­tal pro­to­cols that made iden­ti­fy­ing,
iso­lat­ing, char­ac­ter­iz­ing, and uti­liz­ing genes more ef­fi­cient and rel­a­tively
easy. These tech­no­log­i­cal de­vel­op­ments have had an enor­mous im­pact on
gen­er­at­ing new knowl­edge in prac­ti­cally all­ bi­o­log­i­cal dis­ci­plines, in­clud­ing
an­i­mal be­hav­ior, de­vel­op­men­tal bi­­ol­ogy, mo­lec­u­lar evo­lu­tion, cell bi­­ol­ogy,
and hu­man ge­net­ics. Indeed, the emer­gence of the field of ge­no­mics was
de­pen­dent on the abil­ity to clone large frag­ments of DNA into plas­mids in
prep­a­ra­tion for se­quence de­ter­mi­na­tion.

Commercialization of Molecular Biotechnology

The po­ten­tial of re­com­bi­nant DNA tech­nol­ogy reached the pub­lic with a
frenzy of ex­cite­ment and many peo­ple be­came rich on its prom­ise. Indeed,
within 20 min­utes of the start of trad­ing on the New York Stock Exchange on
14 Oc­to­ber 1980, the price of shares in Genentech, the com­pany founded by
Boyer with chem­ist and en­tre­pre­neur Rob­ert Swanson that pro­duced re­com­
bi­nant hu­man in­su­lin, went from $35 to $89. This was the fast­est in­crease in
the value of any ini­tial pub­lic of­fer­ing in the his­tory of the mar­ket up to that
time. It was pre­dicted that some ge­net­i­cally en­gi­neered mi­cro­or­gan­isms
would re­place chem­i­cal fer­til­iz­ers and oth­ers would eat up oil spills; plants
with in­her­ited re­sis­tance to a va­ri­ety of pests and ex­cep­tional nu­tri­tional
con­tent would be cre­at­ed; and live­stock would have faster grow­ing times,
more ef­fi­cient feed uti­li­za­tion, and meat with low fat con­tent. Many were
 The Development of Molecular Biotechnology 5

Table 1.1 Selected de­vel­op­ments in the his­tory of mo­lec­u­lar bio­tech­nol­o­g y

Date Event Date Event
1917 Karl Ereky coins the term bio­tech­nol­o­gy 1996 Recombinant pro­tein eryth­ro­poi­e­tin ex­ceeds $1 bil­lion in
1940 A. Jost coins the term ge­netic en­gi­neer­ing an­nual sales
1943 Penicillin pro­duced on an in­dus­trial scale 1996 Complete DNA se­quence of all­the chro­mo­somes of a
1944 Avery, MacLeod, and McCarty dem­on­strate that DNA eu­kary­otic or­gan­ism, the yeast Saccharomyces cerevisiae,
is the ge­netic ma­te­ri­al is de­ter­mined
1953 Wat­son and Crick de­ter­mine the struc­ture of DNA 1996 Commercial plant­ing of ge­net­i­cally mod­i­fied crops
1961 The jour­nal Biotechnology and Bioengineering is 1997 Nuclear clon­ing of a mam­mal—a sheep—with a dif­fer­en­ti­
es­tab­lished ated cell nu­cle­us
1961–1966 Entire ge­netic code de­ci­phered 1998 FDA ap­proves first an­ti­sense drug
1970 First re­stric­tion en­do­nu­cle­ase iso­lat­ed 1999 FDA ap­proves re­com­bi­nant fu­sion pro­tein (diph­the­ria
tox­in–in­ter­leu­kin-2) for cu­ta­ne­ous T-cell lym­pho­ma
1972 Khorana and co­work­ers syn­the­size an en­tire tRNA gene
2000 Arabidopsis ge­nome se­quenced
1973 Boyer and Co­hen es­tab­lish re­com­bi­nant DNA tech­nol­o­gy
2000 Monoclonal an­ti­bod­ies ex­ceed $2 bil­lion in an­nual sales
1975 Koh­ler and Mil­stein de­scribe the pro­duc­tion of mono­clo­
nal an­ti­bod­ies 2000 Development of “golden rice” (pro­vi­ta­min-A-producing
rice) an­nounced
1976 First guide­lines for the con­duct of re­com­bi­nant DNA
re­search is­sued 2001 Human ge­nome se­quence is pub­lished
1976 Techniques de­vel­oped to de­ter­mine the se­quence 2002 Complete hu­man gene mi­cro­ar­rays (gene chips) com­mer­
of DNA cially avail­­able
1978 Genentech pro­duces hu­man in­su­lin in E. coli 2002 FDA ap­proves first nu­cleic acid test sys­tem to screen
1980 U.S. Supreme Court rules in the case of Diamond vs. whole blood from do­nors for HIV and HCV
Chakrabarty that ge­net­i­cally ma­nip­u­lated mi­cro­or­gan­ 2004 Large-scale se­quenc­ing of the Sargasso Sea metagenome
isms can be pat­ent­ed 2005 NCBI an­nounces 100 gigabases of nu­cle­o­tides in GenBank
1981 First com­mer­cial, au­to­mated DNA syn­the­siz­ers sold se­quence da­ta­base
1981 First mono­clo­nal an­ti­body-based di­ag­nos­tic kit ap­proved 2006 Recombinant can­cer vac­cine avail­­able to pro­tect against
for use in the United States cer­vi­cal can­cer
1982 First an­i­mal vac­cine pro­duced by re­com­bi­nant DNA 2009 FDA ap­proves first drug pro­duced in a ge­net­i­cally
meth­od­ol­o­gies ap­proved for use in Eu­rope en­gi­neered an­i­mal (goat)
1983 Engineered Ti plas­mids used to trans­form plants 2009 First clin­i­cal trial us­ing em­bry­onic stem cells
1988 U.S. pat­ent granted for a ge­net­i­cally en­gi­neered 2010 Researchers cre­ate the first syn­thetic cell
mouse sus­cep­ti­ble to can­cer 2013 U.S. Supreme Court rules that iso­lated genes are not el­i­gi­ble
1988 PCR method pub­lished for pat­ent­ing
1990 Approval granted in the United States for a trial of hu­man 2014 FDA ap­proves re­com­bi­nant drug pro­duced in the
so­matic cell gene ther­a­py mam­mary glands of trans­genic rab­bits
1990 Recombinant chymosin used for cheese mak­ing in the 2014 U.S. state of Ver­m ont passes leg­is­la­tion re­quir­ing
United States the la­b el­ing of ge­n et­i­c ally mod­i­f ied food in­gre­di­e nts
1994–1995 Detailed ge­netic and phys­i­cal maps of hu­man chro­mo­ 2015 FDA ap­proves first trans­genic an­i­mal (salmon) for hu­man
somes pub­lished con­sump­tion
1994 FDA an­nounces that ge­net­i­cally en­gi­neered to­ma­toes are 2015 Genetically en­gi­neered crops are grown in 28 coun­tries on
as safe as con­ven­tion­ally bred to­ma­toes 180 mil­lion hect­ares
1995 First ge­nome se­quence of a cel­lu­lar or­gan­ism, the 2016 NIH ap­p roves a pro­p osal to test the safety of the
bac­te­rium Haemophilus in­flu­en­za CRISPR ge­n ome ed­i t­i ng tech­n ol­o gy in a clin­i­c al tri­a l
FDA, Food and Drug Administration; HCV, hep­a­ti­tis C vi­rus; HIV, hu­man im­mu­no­de­fi­ciency vi­rus; NCBI, National Center for Biotechnology Information; NIH,
National Institutes of Health; PCR, po­ly­mer­ase chain re­ac­tion; tRNA, trans­fer ri­bo­nu­cleic ac­id; CRISPR, clus­tered reg­u­larly in­ter­spaced short palindromic re­peats.

con­vinced that as long as a bi­o­log­i­cal char­ac­ter­is­tic was ge­net­i­cally de­ter­

mined by one or a few genes, or­gan­isms with novel ge­netic con­sti­tu­tions
could be read­ily cre­ated. Today, in many cases, the prom­ise of re­com­bi­nant
DNA tech­nol­ogy has be­come a re­al­i­ty.
In the 35 years since the com­mer­cial pro­duc­tion of re­com­bi­nant hu­man in­
su­lin, more than 300 new drugs pro­duced by re­com­bi­nant DNA tech­nol­ogy
have been used to treat over 300 mil­lion peo­ple for dis­eases such as can­cer,
6 Chapter 1

Molecular Microbiology Biochemistry Immunology Genetics Chemical Cell biology

biology engineering

Molecular
biotechnology

Crops Drugs Vaccines Diagnostics Livestock

Figure 1.1 Many sci­en­tific dis­ci­plines con­trib­ute to mo­lec­u­lar bio­tech­nol­ogy, which

gen­er­ates a wide range of com­mer­cial prod­ucts.

mul­ti­ple scle­ro­sis, rheu­ma­toid ar­thri­tis, cys­tic fi­bro­sis and strokes, and to

pro­vide pro­tec­tion against nu­mer­ous in­fec­tious dis­eases. The ma­jor­ity of
these are ther­a­peu­tic mono­clo­nal an­ti­bod­ies, hor­mones, and growth fac­tors,
many of which are more ef­fec­tive and have fewer side ef­fects than other
ther­a­pies. Moreover, hun­dreds of new bi­o­log­i­cal drugs are in the pro­cess of
be­ing tested in hu­man clin­i­cal tri­als to treat var­i­ous can­cers, au­to­im­mune
dis­eases, and in­fec­tious dis­eases. Similarly, many new mo­lec­u­lar bio­tech­
nol­ogy prod­ucts for en­hanc­ing crop and live­stock yields, de­creas­ing pes­ti­
cide use, and im­prov­ing in­dus­trial pro­cesses such as the man­u­fac­ture of
pulp and pa­per, food, en­ergy, and tex­tiles have been cre­ated and are be­ing
mar­ket­ed.
The im­pact on ag­ri­cul­ture has been tre­men­dous. While the global pop­u­
la­tion is ex­pand­ing rap­idly, yield in­creases of all­ma­jor crops have de­
creased due to poor ag­ri­cul­tural man­age­ment prac­tices, de­creased acre­age
of ar­a­ble land, and in­creased re­li­ance on fer­til­iz­ers and pes­ti­cides that di­
min­ish soil qual­ity. To pro­duce more food on less land, 18 mil­lion farm­ers
in 28 coun­tries are now plant­ing ge­net­i­cally en­gi­neered crops on 450 mil­
lion acres of land. These crops are pre­dom­i­nantly soy­beans, corn, cot­ton,
and ca­nola that are re­sis­tant to her­bi­cides and in­sects. The global mar­ket
value of ge­net­i­cally mod­i­fied crops is cur­rently $15.3 bil­lion. Small re­source-
poor farm­ers are among the ben­e­fi­cia­ries of ag­ri­cul­tural bio­tech­nol­ogy. In a
com­par­a­tive study of small cot­ton farms in South Af­rica, it was found, over
three sea­sons, that the yield of cot­ton from plants that were ge­net­i­cally en­gi­
neered to pro­duce a bac­te­rial in­sec­ti­cide was on av­er­age about 70% greater
than those from nongenetically mod­i­fied plants. Higher yields and re­duced
pes­ti­cide and la­bor costs trans­lated into dou­bled rev­e­nues de­spite the slightly
higher costs of the trans­genic seeds. In In­dia, which is the larg­est cot­ton pro­
ducer in the world, rev­e­nues from in­sect-resistant cot­ton in­creased by $1.6
bil­lion in 2014 com­pared to the pre­vi­ous year.
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Sekkya Prince, established himself in the hill country about Kyaukse,
only thirty miles from Mandalay, and as late as 1889 gave an
immense amount of trouble, setting the military police at defiance for
months, and committing many murders and depredations. He had an
armed following of several hundreds, and several fights took place
between them and the police. Though the dacoits were each time
defeated and scattered, the ground was so difficult for pursuit, that
they could never catch the leader. At length he was taken in the
Shan States, brought to Kyaukse, tried, convicted and hanged. This
is a specimen of the kind of guerilla warfare going on in every district
all over the country at that time.
Another matter, which still further complicated the situation and
gave strength to the forces of disorder, was the sanction which
dacoity had received through the corruption of those high in office in
the Burman Government before we took it over. A British civil officer
of high rank, the commissioner of a division, writes as follows, as late
as the middle of 1889, more than three years after the annexation:—
“The task of reducing my own division to order I find a gigantic
one. The Burman nature is simply saturated with lawlessness, and it
takes the form of dacoity. Since King Mindohn’s death [i.e., from the
accession of King Theebaw in 1878] it is a fact that most of the
official classes in Upper Burma made large incomes by dacoity. Men
high in office in Mandalay actually kept dacoit bohs, and shared with
them loot, or the subsidies which were paid by the villagers for
protection from other dacoits. The dacoit bohs were actually the
governors, and paid some of the mingyees [ministers of state] in
Mandalay regular sums, on condition of being let alone! Each boh
had a large immediate gang or body of men around him, and a militia
at any time available from the villages. We have had to break up this
system of boh government all over Upper Burma, a system which
had been running for the last ten years. The villagers themselves
have become so accustomed to the government by dacoit chiefs,
that they are actually afraid and even unwilling to help in getting rid
of them. It will be admitted that difficulties like these are enormous;
sometimes they seem to be insuperable, and one is often inclined to
despair. We have not only to deal with the thousands of lawless ones
who think we are encroaching upon their rights, but we have to try
and educate the people to believe that these dacoits are not their
rulers, and are not to be so. The villagers do not yet realise this, and
it is this process of education, slow and painful, that impedes us so
terribly in the work of subjugation and pacification. But the progress
made has been very great.”
The following is given as a specimen of the encounters which for
the first two or three years were of constant occurrence. This affair
was perhaps exceptional in the amount of resistance offered, but in
other respects quite usual and ordinary. It is quoted from a
newspaper dated May 1888:—
“On the night of the 21st inst. 400 dacoits, principally Shans, with
people from Mogaung district, under the leadership of Boh Ti, took
up a position outside Mogaung. Lieutenant O’Donnell, Battalion
Commandant, and Lieutenant Elliot, Assistant Commissioner, with
75 Goorkha military police, patrolled outside the fort the whole night.
At 4 a.m. they attacked the dacoits, who held a strong position in a
series of pagodas, which they had fortified during the night. The
dacoits tenaciously held the position, and the consequence was that
a fierce contest ensued, each pagoda being taken in succession.
The last pagoda, when taken, was found to be choked with dead.
The Goorkha police behaved splendidly. Our casualties were 8 killed
and 15 wounded, while 49 dead dacoits were counted, and over 100
were reported as wounded, most of whom escaped. The struggle at
the last pagoda was hand to hand over a four-foot wall, and
bayonets and spears were used. It was here that 6 out of the 8
police killed fell.”
The mention of these fights deserves a place in any record of
those times, for it was through this hard, rough police and military
work—this continuous pounding at the mass of crime and
lawlessness that would not yield to gentler measures—that the land
now enjoys peace and quiet throughout its length and breadth. There
was manifestly no other way of quelling the disorders and curing the
miseries under which the country groaned.
 This was a specimen of the fighting of our Indian military police;
now for a specimen of that of our English soldiers, who also were
incessantly employed in patrolling the country, and often met with
dacoit bands. The instance given here does not by any means stand
alone; similar affairs often occurred at that time. It illustrates the
courage and dash our men have shown throughout this very
laborious and difficult campaign. Often called to go out in very small
parties, they usually carried the day against all odds; and even
when, as in this instance, they met with such an unusual number of
casualties as to debar them from getting the victory, their coolness
and presence of mind have staved off defeat and disaster, and
enabled them to get through so well that the reverse was,
considering the circumstances, as creditable as a victory would have
been.
“On January 14th, 1889, information reached Lieutenant Nugent,
in charge of a small force of the Hants Regiment, that the advanced
guard of a certain rebel prince was stockaded in a village ten miles
away. He at once decided to attack. He marched out with Sergeant
Bevis and 15 privates, preceded by some of the troops, such as they
were, of the Sawbwa of Momeit. On turning the corner of a jungle
path, their stockade was observed with the gate shut, and white flags
(emblematic of royalty) flying at the gate. The dacoits, on seeing our
men, at once began to blow horns and beat tomtoms. Our Burmese
auxiliaries at once made off, firing their weapons in the air.
Nevertheless Lieutenant Nugent and the 16 Englishmen promptly
charged the stockade, 16 against 200! When about thirty yards from
the stockade the dacoits delivered such a heavy and well-directed
volley that 8 out of the 16 were hit. Private Roberts was killed on the
spot, and Lieutenant Nugent himself was wounded. Seeing that
himself and half his party were disabled, and further assault was out
of the question, Nugent gave the order to get the wounded from
under fire and retire. It is at this point that the soldierly qualities of
these men specially appear. The few men who were able had
meanwhile got under cover of a slight inequality in the ground, and
were keeping up a fire on the stockade. While himself assisting
Private James, who was dangerously wounded, Lieutenant Nugent
was again struck a little below the left breast, this time mortally.
 “Sergeant Bevis now took the command, and rallied his small
party round their fallen officer, and seeing that the dacoits, now
emboldened by observing the small number opposed to them, were
coming out at the gate, he ordered his men to fire a volley. This
caused the enemy to retire inside the stockade, and our party was
molested no more. Stretchers were improvised with rifles and
bamboos for Lieutenant Nugent and Private James, the other
wounded managing to walk. The party made a halt at the village
which they had passed marching out; and here the gallant Nugent
breathed his last. By dint of much pressure and promises of reward
Sergeant Bevis obtained assistance from the Sawbwa’s troops to
carry the body and the bad cases to Momeit.”
Sergeant Bevis was much commended for his good management.
He was promoted at once, and received the decoration of the
Distinguished Service Order. Five days after a small force of
Hampshire men and military police surprised and carried the
stockade.
Many were the deeds of valour in this long and trying campaign. A
considerable number of badges of the Distinguished Service Order
were awarded, and of the highest decoration for gallantry in the field
that military men can aspire to, the Victoria Cross, no less than three
were given.
After what has been said about the Burmese ministers of the
Crown, it will be no matter of surprise that the honest attempt of the
British Government to utilise the local knowledge and experience of
the Hloot Daw or supreme council of the king, as the medium of
government, should entirely break down. As might have been
expected, those worthies were found to be worse than useless at
such a crisis. The kind of government they had been accustomed to
administer was just the kind that was not wanted. They were
therefore pensioned off, the pension acting in a twofold manner, as a
substantial compensation for loss of office, and as a guarantee of
their loyalty; they had something to lose.
During the first year or two of the British occupation there was
need for very special vigilance to prevent the carrying out of plots of
insurrection, especially in Mandalay. It was of course childish to think
they could dislodge the British power, but many of the people were
slow to believe this, and foolish enough to listen to boasting
proposals of this kind. However, such a good watch was kept, and
the officials kept themselves so well informed, that all such attempts
were nipped in the bud. Some idea of the magnitude of the work of
pacification may be gathered from a paper published by the Chief
Commissioner of Burma in 1889, from which it appears that no less
than 363 dacoit bohs or leaders were either killed, or surrendered, or
were taken prisoners between April 1887 and August 1889.
The British Government, whilst very stern in pursuing, arresting
and punishing these notorious outlaws, made every concession
towards mercy where it was possible. When a gang of dacoits was
broken up, and the boh killed or taken, the men composing it were
usually allowed to settle down in their villages, giving some sort of
guarantee for their future good behaviour. As soon as it became safe
to show any considerable leniency, the cases of all who had been
sentenced to terms of penal servitude for participating in dacoity
were carefully gone through by an experienced and able judicial
commissioner, for the purpose of remitting the punishment wherever
it could safely be done, particularly in cases where men had been
led, during a time of anarchy and political excitement, to take part in
crimes and acts of violence, from which, under ordinary
circumstances, they would have abstained. The result was that 899
prisoners were set at liberty at once, and 450 more were promised
their release in the following December if their conduct in jail
continued good. Only the worst and most desperate offenders were
kept in jail.
It is just possible that some readers, failing to realise the full force
of all the circumstances, may be inclined to think that the information
given in this chapter leans too much in the direction of admiration of
the military deeds described, and is lacking in consideration for the
case of the unfortunate men against whom these operations were
directed. I feel that it would ill become me to do anything to fan the
flame of the military spirit, for militarism is without doubt one of the
great curses of this age, and I have had no such design in view. I
have merely described what took place. If the reader feels inclined to
admire any of the actions here described, I must give him notice that
he does it entirely on his own responsibility.
It may occur to the reader that perhaps after all it was the spirit of
patriotism that animated these Burmans. Were they not fighting for
their country and their liberty, and doing their feeble best to cast out
the invader? Doubtless there was in some cases something of this
feeling in their minds, enough to give a colourable pretext to their
conduct at the time. But there are considerations that go to show that
if we are to make any allowance on this account it will have to be
very little.
Dacoity existed and was rampant for years prior to our annexation
of the country.
How is the motive of patriotism to be reconciled with the gross
cruelty, and robbery, and murder which all the dacoit bands
continually practised?
When so many hundreds of bohs were fighting, each for his own
hand, which were we to recognise? And how many? Their claims to
the mastery were mutually antagonistic.
I have already said that I decline to take the responsibility either of
defending or of impeaching the action of England in the invasion of
Upper Burma. It involves the great and wide question of Empire,
which I leave to more competent hands. I content myself with giving
the facts from the standpoint of an eyewitness, and enabling or
assisting wiser men to settle the greater question. I take up the
question at this point—England the de facto ruler. Somehow,
rightfully or wrongfully, she is there, and has undertaken the
government of the country. The country is in a flame with crime and
disorder. What is she to do?
There have been times, even in our own country, when certain
crimes of violence, such as garotting, and certain forms of murder,
have spread so as to cause almost a panic, and have needed
special measures both as to detection and punishment. We are far
more liable to such things in India. Take, for instance, that strange
phase of crime known as “thuggee,” which prevailed to a fearful
extent years ago in India, and to which, in respect of each being an
epidemic form of crime, dacoity in Burma has sometimes been
compared. Thuggee was a thoroughly organised system of robbery
and murder, carried out with great secrecy by an association of men
banded together for the purpose, and who did it not by open assault
but by stealthy approaches, and, strangest of all, with religious
motives. The verdict of civilised society was that the extermination of
the thugs was not only a justifiable thing to be done, but the solemn
duty of the Government, notwithstanding the religious motives, and
special officers of Government were deputed for that purpose, and
the system was finally stamped out.
So with dacoity. If men will be brutal, will set all law, human and
divine, at defiance, will make human life cheap and property unsafe,
and keep the whole country in terror and confusion, to the detriment
of all peace and progress, if, in short, they will come to no terms, but
deliberately elect to assume the character of wild beasts preying on
society, then all reasonable men will feel constrained sorrowfully to
admit that a civilised Government has no alternative but to treat them
as such, and hunt them down; always however remembering that, as
it is in the divine, so in the human administration, justice should be
tempered with mercy; and wherever there is room to hope for better
things, the criminal should have another chance, a provision which
our Government, as I have shown, has not neglected.
 CHAPTER VI.
BRITISH INFLUENCE IN THE SHAN STATES.

The previous chapter dealt with the pacification of Upper Burma

proper, that tract of country which England has annexed, and in
which we have assumed the full responsibility of government. In this
chapter we have to consider our relations with certain states and
tribes on our frontiers, which are not British territory, but for whose
well-being and good behaviour we hold ourselves to some extent
responsible, in proportion as our influence among them is more or
less direct.
As soon as our first difficulties in the pacification and
administration of Upper Burma were to some extent overcome, our
Government had to turn its attention to the doings of the many
barbarous and semi-barbarous tribes and races in the regions
immediately adjacent to Burma.
To the east of Upper Burma, and situated between that country
and the great empire of China, are the Shan States tributary to
Burma, with an area about four-fifths that of England, but with a
population no larger than that of Worcestershire, not one-fourth, it is
said, of what it was fifty years ago. This country is a very fine one,
consisting of a great plateau with a diversified climate and great
natural resources, of which coal is one, though it has not yet been
worked, and with every capacity for development. The Shan States
are likely to play no unimportant part in the commercial development
of the next few years, for it is by that route that the railway will go
from Burma to China at no distant date.
At present these states are in a most backward and uncivilised
condition, and as they afford such an interesting illustration of the
true frontier policy of England in the East, and the kind of influence
our country is so well able to exert, in the discharge of her duty as
the great suzerain power amongst many little races and peoples, I
make no apology for describing it with some degree of detail. Such
work as England is attempting to do, and will in the end undoubtedly
succeed in doing there, is so beneficent and meritorious as to be
beyond the possibility of objection; and it would excite remark and
applause if it were not so common—if England were not doing much
the same all over her Eastern dominions.
The relation of the Shan States to the British rule is a feudatory
relation. They paid tribute to the King of Burma, and were supposed
to be subject to him, but although receiving tribute, Burma conferred
no benefits upon them. In fact, the idea that something in the shape
of government was due to the Shans, in return for the tribute they
paid, probably never entered the head of King Theebaw. These
states have not been annexed to British territory, and are not likely to
be, unless it should be found quite impossible to get their chiefs to
learn to rule properly. At present the policy is entirely in the direction
of setting these native rulers on their feet, and strengthening their
power as much as possible. When the English commenced to rule at
Mandalay that feudatory relation to the defunct Burmese
Government passed over to the English.
Politically the Shan States are divided amongst some eighteen
chiefs, each ruling a greater or less extent of territory. In the early
part of 1888 two British expeditions were sent to the Northern and
Southern Shan States respectively, and the first steps were taken
toward adjusting our relations with them.
The condition in which the States were found by the British forces
was a very sad one. For want of a controlling power over them there
was a state of disorder amounting almost to anarchy. Might was
right, and in the struggle for mastery the Shans were fast
exterminating each other. Each petty chieftain with his followers was
on the look-out to extend the sphere of his rule by aggression, and
dacoit raids and incessant civil war were the result.
Throughout the reign of King Theebaw the States had suffered,
and the population had so seriously fallen off, by war and perhaps
too by emigration, that land had fallen out of cultivation, and
prosperous towns had been reduced, in some cases, to one-tenth of
their former size. Added to this there had been a season of scarcity,
and cattle disease had been very fatal.
The people cordially welcomed the advent of a strong power that
could enforce peace amongst them; and what was wanting for the
temporal salvation of this distressed country was just that kind of
sovereignty and paternal rule which England was able and willing to
give them. It was necessary for England to assert and maintain her
rights as the suzerain power, and to discharge her duties by taking
them under the broad shield of her protection and guidance.
The British representatives accordingly received the personal
submission of all the principal sawbwas or chiefs, confirmed them in
their positions as tributary rulers, settled their relations with
Government and with each other, fixed the amount of tribute to be
paid by each chief, and succeeded in placing the administration of
the states on a satisfactory footing. Two British officials were
appointed as Superintendents of the two divisions of the Shan
States, northern and southern. Tribal disputes were henceforth to be
referred to these officials for arbitration, and fighting between
individual states was strictly forbidden. They were not to enter into
relations with any other foreign power; and they were gradually to
approximate their primitive methods of government to our standards.
In return for these conditions, to be fulfilled by the Shans, certain
very substantial advantages were bestowed upon them by the
British. Each chieftain is recognised and protected in the exercise of
his chieftainship.
The import duties formerly levied by Burma on goods going from
Shanland into Burma are abolished, to the great advantage and
encouragement of their trade.
The great want of means of communication through the country is
being met by the construction of roads by the British Government, at
its own expense.
A preliminary survey has been made of the different routes for a
railway to run through the country, and a more accurate and detailed
survey of the one chosen is to be made shortly.
The navigation of the upper parts of the Salween River, which
flows through the Shan States, is receiving attention with the view of
utilising it for purposes of trade, if it be found practicable.
Experiments are being made under the auspices of the British in
the Shan country, in order to introduce the cultivation of new cereals
and other products amongst them, and to improve their breed of
cattle and sheep.
In short, England is trying to do her duty by this naturally
magnificent but very backward country, and it may be confidently
stated that if any Government could help them on their feet it is the
one they now have. The most recent information from the Chief
Superintendent of the Shan States, the responsible British officer
appointed to look after them, shows that he finds them in a most
benighted and backward condition socially and politically, and there
will be need for lengthened intervention and much patience and
perseverance on the part of the British Government. It is found that
there has been no such thing as law in the country, written or
unwritten. Everybody does what is right in his own eyes, if he can.
The hold which these chiefs have on the territories they are
supposed to rule is of the feeblest description; and it will require time
for the people to get out of that state of turbulence, unrest and
distraction, and for the rulers to acquire power and experience for
civil rule. Like incompetent rulers, they try at present to maintain their
authority by inflicting most barbarous punishments for the most trivial
offences.
The Sawbwa of Thibaw is reported to be the only chief among
them who exercises any real and active control throughout his state,
and he endeavours to enforce the rule that the power of awarding
capital punishment shall be restricted to the chiefs. In all the other
states the people are fleeced by the minor officials, and criminal
justice is administered in a cruel and haphazard fashion. An English
traveller recently found the fresh head of a so-called thief posted up
in the Mangko bazaar; and in another place through which he
travelled a boy of sixteen was summarily killed and barbarously
mutilated, on the ground that he had been seen entering a buffalo
shed, and was therefore supposed to be attempting cattle-stealing.
As a beginning in the way of much-needed reform, our paternal
Government has framed for their guidance a few simple rules for the
administration of criminal justice, and supplied them to each
chieftain, as a sort of alphabet of government for them to learn. I
wonder what they think of our notions of justice. They must appear to
them unaccountably and unnecessarily lenient towards the prisoner.
How it must puzzle them, for instance, to be told that an accused
person must be presumed to be innocent until he is proved to be
guilty!
As a lesson in revenue and finance, each chief is now required to
frame a simple form of budget for his state, subject to the approval of
the Superintendent, fixing the amount to be devoted to the private
expenditure of the ruling family, and making reasonable provision for
the administration of civil and criminal justice, police, and public
works. It puts one in mind of a class of boys coming up with their
lessons written out for the teacher to see; but it is evidently needed
work, and it will not do to despise the day of small things. It will of
course be a new idea to them that anybody else but the sawbwa
himself has anything to do with the expenditure of the revenue of the
state, which they have always been accustomed to consider as his
private property. But Orientals take kindly to this tutelage, and will
scarcely think of resenting it, though they might be tempted to
neglect it if they could. And it must not be supposed that this case of
the Shan States is any rarity, for this kind of inspection, instruction
and guidance is only what we are called upon to do in a greater or
less degree in all the protected states which are feudatory to our
Indian Empire, and in other parts of the world.
The Chief Commissioner of Burma, to whom all the chiefs are
amenable, commenting on the above rules, endorses the opinion
expressed by one of the Superintendents, that it will probably be
found impossible to effect any real reforms until a trained Dewan
(Prime Minister) is appointed for each state to teach the rulers how
to rule. As England is very resolute in all she takes in hand in this
way, perhaps in course of time some faint sense of the responsibility
of ruling may find its way into the minds of these benighted Shan
sawbwas. But if it be not so, and if in the end England should find
herself compelled, in the interests of humanity, to take a still larger
share of the responsibility of ruling in that country, of which however
there is at present no sign or mention, the foregoing information
clearly shows that it will not be for want of an honest effort to get
them to do it themselves.
All this explains incidentally how it is that Empire with its
responsibilities grows on our hands. In human affairs, when a man
does his work well, you promote him by giving him more work to do.
When the sudden emergency arises men naturally saddle the willing
horse. It is so throughout the divine economy also. “Whatsoever thy
hand findeth to do, do it with thy might.” “For unto every one that
hath shall be given, and he shall have abundance: but from him that
hath not, even that which he hath shall be taken away.”
Of one thing there is no doubt, the states now enjoy tranquillity
and the beginnings of prosperity such as they have not enjoyed for
many years. Not long ago, meeting a Shan who had just come to
Mandalay several days’ journey on foot through the Shan States, I
asked him what was the present condition of the country. His reply
was, “So quiet, that even an unprotected female could walk through
it.”
The chieftain mentioned above with approval as an exceptional
prince, and more enlightened than his fellows, is the Sawbwa of
Thibaw. He once had a curious experience, that appears to have
considerably opened his mind and enlarged his ideas. Some years
ago, before the annexation of Upper Burma was even thought of, he
paid a visit to the great city of Rangoon. Like the Queen of Sheba,
who had heard of the wisdom and glory of Solomon, he had received
tidings of the great transformation that had taken place in that city,
and wished to see the British power for himself. Possibly, as the
Shans are Buddhists, he might be inclined also to pay a visit to the
world-renowned Buddhist shrine at Rangoon, the Shwê Dagohn
pagoda. To venture so far away from his remote inland state among
the mountains shows him to be a man of some natural force of
character, for most sawbwas would have been afraid to leave their
states for so long. Whilst in Rangoon one of his retainers displeased
him, and in a burst of anger he killed him on the spot. But,
unfortunately for him, this had happened in British territory, where
they call such actions, no matter who does them, by the name of
murder; and he was accordingly arrested and put in jail to stand his
trial for that crime. His plea was of course that he was a king, and
that he had the power of life and death; and seeing that such was
the case in his own territories, and that he had no idea he was
exceeding his prerogative in doing as he did, he was released, and
some good advice was given him for future use. It is gratifying to find
that this experience has borne fruit, and that years after, when in
course of things the Shan States have become tributary to Britain,
and an attempt is being made to bring them somewhat into line with
more enlightened nations, he is officially named as the most
progressive and reliable of the Shan rulers.
Other operations for the pacification of our Burmese frontiers may
be mentioned here. Amongst the barbarous and unlettered tribes on
the mountains in the north there has been a continuance of the kind
of lawlessness prevalent in the days before our rule in Upper Burma.
The tribes of wild Kachins there have given considerable trouble
from time to time. They are warlike and predatory, and in their
mountains and jungles able to offer considerable resistance.
Occasionally, too, in the north, large numbers of disbanded
Chinese soldiers have turned dacoits, and crossed the frontier into
the Bhamo district to plunder. They have, however, suffered severely
whenever they have tried conclusions with the British columns sent
out against them. Attention is being given to the delimitation of the
Chinese frontier, which will lead the way to a better protection of it on
both sides. In the east the Red Karens gave trouble, while on the
west the wild Chins of the Arakan Yoma mountains continued their
former practice of raiding into Burma and carrying off loot and
captives.
THE GOLDEN PAGODA AT MANDALAY, COMPLETELY COVERED WITH GOLD
LEAF.

All this had to be brought to an end, and these lawless marauders

given clearly to understand that it would no longer be permitted, but
that a power now ruled in Burma that was able to keep them in
check, and would protect the interests of its subjects against their
acts of rapine and violence. Several expeditions were undertaken for
this purpose to the different mountain tribes, and much hard, rough
work had to be done; but beyond keeping these tribes in order in
relation to Burma, it is uncertain yet what measures England will
initiate for their internal government.
In connection with these different expeditions much valuable
exploration and surveying work have been done on our frontiers, in
what was formerly an unknown country.
On the whole, it will be seen that to restore order and establish
good government, in a country like this, and under such
circumstances, was a work of gigantic difficulty, requiring much
activity and vigilance, much firmness and courage, readiness of
resource, and withal a long purse. What has been spent, however,
may be regarded as capital well laid out, that has already begun to
be productive. Seldom, perhaps, has England undertaken a heavier
task so far away from home; never has she accomplished it with
more credit. Gradually, but surely, the British talent for organising
and ruling has asserted itself, and the great resources at our
command, despite the smallness of our numbers on the spot, have
materially helped to win the victory. One cannot but admire that
splendid courage, and that administrative ability, whereby our
countrymen have taken over a country of vast extent, in a condition
bordering on anarchy, and in five years, with the aid that India has
been able to give in men and means, they have made it safer and
more prosperous to live in than at any previous period of its history in
modern times.
The more extended notice of the progress made in the material
development of Upper Burma is reserved for another chapter.
 CHAPTER VII.
FIVE YEARS OF BRITISH RULE.

British rule would have nothing to justify its presence in such a

country as Burma if it did not evidently make for the well-being of the
people. In this chapter we have to consider the initiation of those
measures that have been adopted with this view, and to ascertain
how far they are likely to secure it. Five years is not a period of time
from which much can be expected by way of results, but it is long
enough for us to form an estimate of the kind of beginning that has
been made.
Under Burmese rule no attempt was made at a division of the work
of the executive into departments. Each minister of state was
considered eligible to take charge of any and every post in the state,
whether judicial, revenue, military or what not, just as in England, as
Macaulay tells us, until comparatively recent times, any gentleman, if
he possessed sufficient interest, might aspire to command a man-of-
war, and naval and military commands were more or less
interchangeable. But we have got far beyond that now, and our
Indian Government is a model of efficiency and business-like
working, the officers of some departments being professionally
educated for them, and in others, specially trained for the work.
The state of the public revenue is always some test of the
industrial and fiscal conditions of a country. Beginning with the first
year of the annexation, the income for the five years has steadily and
rapidly risen:—

Rupees.
In 1886-87 the revenue was 2,200,000
1887-88 ” ” 5,010,000
1888-89 ” ” 7,683,450
 1889-90 ” ” 8,638,170
1890-91 ” ” 9,400,000

To the amount for the last year a considerable sum might fairly be
added on account of the earnings of the new line of railway to
Mandalay. Under the Burman king the revenue never exceeded
10,000,000 rupees, and during King Theebaw’s reign it had fallen to
9,000,000, and fully one-third of this amount accrued from
monopolies and imposts on trade and industry, that the British
Government has very properly abolished; so that, although we took
over the country at a very great disadvantage, we have already
raised the revenue, by healthy and legitimate means (excepting the
excise), to an amount equal to what it ever was before. There can be
no doubt that a career of prosperity awaits Upper Burma, and that
the steady increase in the revenue indicates that it has already
entered upon that career. The testimony of the revenue officers is
that it is, as a rule, collected without difficulty, and that the taxation
does not fall at all heavily on the people. The chief item is a kind of
capitation or household tax, averaging 10 rupees per house per year.
This is levied as a lump sum on each village, and the payment is
distributed amongst the families of the village, according to their
means and circumstances, by a committee of village elders—a
method they are accustomed to, and that seems to work well.
The administration of justice is one of the fundamental duties of
Government and one of its chief functions. Our Government
undertook this duty amid special difficulties and drawbacks; for not
only were crime and disorder very general, but there was a great
paucity of officials with the necessary experience of the country and
knowledge of the language, to fill the subordinate grades of the Civil
Service, and to act as magistrates. It must have been no easy task
to administer justice at once over an area as large as France. Great
progress has been made during the five years, and the various
courts of justice have long been in good working order after the
methods of India.
The adaptation of a regular system of criminal law, as laid down in
the Indian Penal Code, with British principles as regards evidence