Gene Biotechnology 9789350432570, 9788184885606

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GENE BIOTECHNOLOGY

S.N. JOGDAND Dept. of Microbiology, Karma veer Bhaurao Pati! Col/ege, Vashi, Navi Mumbai.

ISBN :978-81-84885-60-6 (

I

REVISED EDITION: 2009 )

Hal Glfimalaya GpublishingGlfouse MUMBAl • NEW DELHI • NAGPUR • BANGALORE • HYDERABAD • CHENNAI • PUNE • LUCKNOW • AHMEDABAD • ERNAKULAM

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Revised Edition

Published by

2009

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CONTENTS

I ~

1

From Classical Genetics to Modern Genetics

2

Genetic Engineering and r-DNA Technology

3

Genetic Complexity

4

Enzymes used in Gene Manipulation of DNA and RNA

24

5

Restriction Endonucleases-Molecular Scalpels

31

6

Isolation and Purification of DNA

7

Plasmids

41

8

Isolation and Purification of Plasmid DNA

49

9

Preparation of RNA

55

10

Autoradiography

59

11

Electrophoresis of DNA

12

Pulsed Field Gel Electrophoresis (PFGE)

13

Nucleic Acid Staining

70

14

Nucleic Acid Labelling

79

15

Molecular Probes

83

16

Hybridization Techniques

89 '

17

DNA Fingerprinting (DNA Profiling)

99

18

Restriction Fragment Length Polymorphism (RFLP)

108

19

Slotting Techniques

111

20

Vectors

116 I

21

Gene Cloning

138

22

Screening of Recombinants

151

23

Protoplast Technology - Fusion Techniques

159

24

DNA-Protein Interactions

167

25

DNA Libraries (a) Genomic Library (b) cDNA Library

172

~

i i

-.

I

I

37

I i

62 I

70

I I

~

I

l

1

26

Chromosome Walking and Jumping

181

27

Site-Directed Mutagenesis

187

28

DNA Synthesis

193

29

Techniques of (Chromosome) Gene Mapping

198

30

DNA Sequencing (Gene Sequencing)

208

31

Ribozymes

215

32

Polymerase Chain Reaction (PCR)

221

33

Random Amplification of Polymorphic DNA (RAPD)

233

34

Subtraction Hybridization

237

35

Ribotyping

240

36

Protein Engineering

245

37

Strain Imr.;rovement of Industrially Important Organisms

256

38

Gene Transfer Technologies (DNA Transfer Methods)

263

39

Transgenic Animals

276

40

Human Gene Therapy

286

41

Gene Targeting

295

42

Antisense Therapy

301

43

RNA Interference

307

44

Flow Cytogenetics

312

45

Human Genome Project - Part I

319

45

Human Genome Project - Part II

328

47

Genomics

336

48

Proteomics

341

49

Bioinformatics - I (Primer)

352

50

Bioinformatics - II (Tools and Techniques)

365

51

Ethical, Legal, Social, Environmental and Health Issues Related to Gene Biotechnology

376

52

Sythetic Biology

387

Glossary

400

References

406

Module 1

FROM CLASSICAL GENETICS TO MODERN GENETICS 1.1

Introduction.

1.2 1.3

Landmarks in molecular biology and biotechnology. Advantages of using microorganisms for genetic research.

1.1

Introduction Genetics is the fundamental biological science. Central to the principle of genetics is the concept of heredity. Full understanding of biological process is possible only with detailed analysis of gene structure and function. Classically, this was done by study of mutants - study of properties of mutants, mapping and generating hypothesis. The main principle used by classical genetics was - the function of genes and their products would be established if we can observe what happens when the genes are mutated. There were many problems and limitations of classical genetics (1) (2)

(3) (4)

(5)

Major limitation was that only mutations that produce readily detectable phenotype can be studied. Other problem is that in diploid organisms many mutations can be detected after appropriate breeding strategies to generate homozygotes tor mutation. This means many mutations may be missed and with animals, breeding cycles will take months or years. Man cannot be subjected to genetic analysis except by retrospective studies of family pedigrees. Some mutations are lethal in homozygotes. Many key steps in early embryogenesis and differentiation fall into this category and cannot be studied in depth by classical genetics. Existence of a gene is proved only by discovery of mutation in a gene. Genetic maps are not established by studying normal genes but is possible by locating the mutant forms (alleles). Any gene for which mutant or variant has not been detected, officially does not exist according to classical genetics.

Mutation still remains an important essential tool for 'geneticist.' However, now mutations can be introduced more at will and at predetermined location.

(1)

2

Gene Biotechnology

Method of mutation is restricted to unicellular organisms or cultured ceils of higher organisms but not on multicellular complete organism, plant or animaL Classical genetics, more worked to understand basics of fleredily. It was rather a stage of learning natural phenomena and peeping a bit into mechanisms involved. It was only a beginning of scientific approach in study of life processes. 'Genes as the units of heredity' was comparatively a new finding. The existence of RNA and DNA as the biochemical base of heredity was also new. And although mutational programmes were exercised, structure of DNA. understanding of genetic code, DNA replication were simultaneously studied. Most of the part of first half of twentieth century we were still busy in observing nature with aim of reaching to the core principle of heredity. Natural phenomena of genetiC material transfer like transformation, conjugation were understood in next few years. As the other sciences were also not greatly developed instrumentation available had limitations. It is difficult to ascertain as to which discovery was the turning point, as to which discovery took us from a stage of understanding of basic prinCiple to applying it to our benefit and as to which discovery has brought us to regulation, control and speed and wilful gene manipulation stage. But the progress in the last 25-30 years has been tremendously rapid. Classical genetiCS is being replaced by a technological stage-molecular biology and biotechnology. The progress in instrumentation, the discovery of engineering techniques, the advent of computerization has taken us at more commanding stage in study and exploitation of life processes. Improvements in gene transfer technologies has enabled our progress in interest of exploiting the nature. Genetic engineering, recombinant DNA technology. gene cloning, gene targeting, gene therapy and all such terminologies only suggest our command on life's basic units. We may refer to technological stage of genetiCS as the modern genetiCS just to put a distinction from classical genetiCS. The aims and objectives remain the same. only the achievements have been remarkable and dimension of progress is astonishing. Some of the contributions of modem genetics are: (1) (2) (3) (4)

(5) (6) (7) (8)

Methods of transferring cloned DNA back into living cells has provided the key to understanding a vast spectrum of fundamental biological prOG9Sses. Producing gene products (e.g., hormones and interferons) in large quantities in cells which are simple and easy to cultivate. Isolation and charaterization of genes involved in cancer. Precise models of human diseases (such as cancer, diabetes, muscular dystrophies, growth defects) are established by introducing new genes and gene combinations in mice. It has revolutionized the approaches of genetic analysis. All possible mutations can be studied by simply changing cloned DNA before reintroducing it into cells. Interspecific gene transfer to establish conservation or divergence of biological processes. Fundamental processes such as DNA replication and chromosome behaviour can be studied because key DNA components have been artificially reconstructed. Complex processes of development and differentiation can be analysed. Regulatory interactions can be studied by introducing different combinations of genes into same cell and directly testing their interactions. This can be done with :;impl9 culture systems that generate answers in weeks rathei than in years.

Apart from few contributions mentioned 3.bove, modern genetics has its impact on every aspect related to exploitation of life processes. Ample food, cheap energy, healthy disease free

3

From Classical Genetics to Modem Genetics

animals, high yielding disease resistant, pesticide resistant plants, improved analytical tools (DNA probes, monoclonal antibodies) protection of human against infectious diseases, control of genetic disorders, production of chemicals, vitamins, etc. in safer and cheaper way, cleaner environment, are the various areas of achievements for which modern genetics can claim its big share. The transformation from classical genetics to modern genetics has been rapid, impressive and fruitful. It is difficult, to keep pace with this fast development. Next few modules in this book are only a humble attempt to cover some aspects of this modern genetics.

1.2 Some Landmarks in Molecular Biology and Biotechnology Year Development

Contributor

1865 1920

Genes as the units of heredity Two kinds of nucleic aCids (RNA & DNA)

Gregor Mendel Levine & Jones

1928

Transformation of non-pathogenic Diplococcus pneumonia by mixing with killed pathogenic D. pneumoniae

Fred Griffith

1917 1943

Term biotechnology comed by Resistance to phage infection arises from spontaneous mutation

Karl Ereky S.E. Luria, M. Delbruck

1944

Transforming factor in Griffith's experiment as DNA

Avery etal.

DNA is the genetic material

Avery Macleads McCarty

1946

Bacterial recombination discovered

1951

Lambda bacteriophage discovered

1952

Host controlled restriction - phage growth restricted on certain strains

J. Lederberg, E.L. Tatum J. Lederberg S.E. Luria Bertani

·1944

1952 1953

Plasmid term for oxtrachromosomal DNA F factor discovered -- the first plasmid shown also to be responsible for conjugation

Lederberg W. Hayes

1953

Double helix model of DNA

Watson & Crick

1956

Integrated F plasmid (Hfr) strains used for large-scale mapping of bacterial chromosome

F.•Jacob

1956

Isolation of DNA polymerase I, first enzymatic synthesis of DNA

1957

First biologically aCiive DNA synthesised - coliform phage x 174

1958

Semiconservative replication of DNA shown

1959-62 1961 1961

Fine structure genetic map of rllA and rllB genes of coliform T 4 developed Nucleic acid Hybridization Operon model for genetic regulation of protein synthesis

1961

In-vitro protein synthesis allows codon assignations

1961-66

The entire genetic code deciphered

1966 1966-68

First chemical synthesis of a gene DNA restriction enzyme isolated from strain K

1966 1967

Micro-injection oj mouse eggs Classical method of plasmid isolation in pure state

E.L. Wollman W. Hayes A. Kornberg A. Kolberg R.C. Sinbeimer M. Meselson FW. Stahl S. Benzer Marmur and Doty F. Jacob J. Monod M.W. Nirenberg H. Matthaei H.G. Khorana M. Meselson T.P. Lin Vinograd

4

Gene Biotechnology

Year

Development

Flow cytogenetics method developed Nomenclature of restriction enzymes First recombinant plasmids based on the coliform vector pSc 101, transform E. coli. (established r-DNA technology) Flow Karyotype derived from human cell line 1975 1975 Southern blotting 1979 Northern blotting for RNA Production of monoclonal antibodies described 1975 Colony hybridization 1976 First guidelines for conduct of recombinant DNA research issued 1976 Technique developed to determine sequence of DNA 1976 1977,80 DNA sequency (first method) DNA sequencing method 1977 Plaque hybridization 1977 Cosmids first developed 1978 1970 1973 1973

1978

Genetech produced human insulin in

1978

Fusion of sphaeroplasts of yeast and transformation done for the first time Current most popular method of extraction and purification of plasmid DNA Micro-injection of fertilized eggs Site directed mutagenesis U.S. Court, ruling in favour of patenting of genetically engineered organism of Dr. Anand Chakrabarty First monoclonal antibody based diagnostic kit approved for use in United States First commerciai automated DNA synthesizer sold First animal vaccine produced by recombinant DNA methodapproved for use in Europe Pulsed Field gel electrophoresis used for the first time Engineered Ti plasmids used to transform plants Polymerase Chain Reaction (PCR) Chromosome jumping Development of original probes to identify 'minisatellites' in DNA finger printing Biolistic transformation First RFLP map of human genome Use of liposomes for DNA transfer 'Antisense' experiment on Petunia U.S. Patent granted for genetically engineered mouse susceptible to cancer Approval granted in United States for trial of human somatic cell gene therapy First gene therapy on human patient of SCID case

Contributor Wray & Stubblefield Smith & Nathans H.w. Boyer S.N. Cohen Grayetal. E.M. Southern Alwine, Kemp, Stark Kohler & Milstein Grunstein & Hogness

Maxam and Gilbert Sanger Benton & Devis Barbara Hohn, John Colhns

E. coli.

1979 1980 1980 1980 1981 1981 1982 1982 1983 1983 1984 1985 1987 1987 1988 1988 1988 1990 1990 1990

Human Genome Project Launched as coordinated effort of U.S Department of Enerav and the National Institutes of Health.

Hinnen et al. Bimboim and Doly Gorden et al. Gillam etal.

Schwartz et al. Kary Mulis Collins & Weissman Alec Jaffrey Sanford et al. Donnis-keller et al. Paphdjopopulos Van DerKrol

Michaele Blease and W. French Andrescow

From Classical Genetics to Modem Genetics Year 1990

1990's 1990's 1993 1993

Development First gene successful therapy in humans (in ADA deficiency causing SCID); the California Hereditary Disorders Act comes into force; human genome project begins DNA microarrays are invented DNA fingerprinting, gene therapy, and genetically modified foods come onto the scene. Huntin~ton's disease gene is identified; gene therapy for SCID and cystic fibrosis begins in the UK Finding a major susceptibility gene for the late-onset form of Alzheimer Disease.

5 Contributor WF Anderson in the USA Pat Brown and colleagues

Allen Roses, MD, and his colleagues at Duke University

1994

·The FlavrSavr tomato is approved by the FDA as the first GM food to go on the market (now discontinued) 1994 A high-density genetic map of the human genome, consisting of almost 6,000 markers, is published in Science magazine. 1995 The genome of Haemophilus influenzae is the first genome of a free living organism to be sequenced 1996 Saccharomyces cerevisiae is the first eukaryote genome sequence to be released 1996 The cloned sheep" Doily" is presented 1997 Complete E. coli genome is sequenced 1998 Human genetic map is produced, showing the chromosomal locations of markers from more than 30,000 human genes. 1998 Caenorhabditis e/egans becomes the first multicellular eukaryote whose genome is totally sequenced 1998 University of Hawaii scientists clone three generations of mice from nuclei of adult ovarian cuulus cells. Human skin is produced in vitro. Embryonic stem cells are used to regenerate tissue and create disorders mimicking diseases. 1999 A human MHC (HLA-DR52) haplotype is totally sequenced (October). Human chromosome 22 becomes the first one to be sequenced completely. 2000 Completion of Arabidopsis thaliana sequence and Drosophila sequence 2001 First draft sequences of the human genome are released simultaneously by the Human Genome Project and Celera Genomics. 2001 A private U.S. research company Advanced Cell Technology (ACT) announces it has cloned human embryos. The company says the intention is not to create cloned human beings but to make lifesaving therapies for a wide range of human diseases. Political and religious leaders around the world condemn the effort. 2002 The first cloned pigs genetically modified specifically for the purposes January of replacement human organ transplantation were born at the Roslin Institute. 2003 Successful completion of Human Genome Project with 99% of the (24th genome sequenced to a 99.99% accuracy. April) 2003 Complete sequence of human V-chromosome is published 2007 Controversies continue over human and animal cloning, research on stem cells, and genetic modification of crops.

Wilmut and colleagues

6

Gene Biotechnology

1.3

Advantages of Using Microorganisms for Genetic Research Importance of microorganisms in genetic research became apparent when difficulties were faced in study of biosynthetic (metabolic) pathways with the then popular candidate for researchthe fruit fly-Drosophila. This was in 1930s. Understanding the difficulties faced, will help to realise advantages of microorganisms (1) (2) (3) (4)

Fruit fly Drosophila though had generation time of 14 days (relatively short) breeding of enough of them for chemical analysis was still difficult. Direct comparison of chemical composition of mutant and wild type was not possible. Fruit flies are diploid, therefore, most mutations may go undetected if they are recessive. Multicelied organisms are relatively difficult for genetic studies.

Then single celled eukaryotic alga like Chlamydomonas, the yeast Saccharomyces Cerevisiae and the mould Neurospora Crassa were amongst the first subjects of genetiC analysis and experimental studies. Advantages ,were: (1) (2)

They were haploid. They multiply rapidly in less than two hours of generation time.

Upto 1940s, Neurospora crassa was popular experimental organism and initial work on one gene-one enzyme was successfully done with this organism. In those days, detection and differentiation between bacteria was not possible except for using the criteria of colony characteristics on solid media. Later, detection of variants of bacteria on solid media using their antibiotic resistance or phage resistance properties and development of replica plate technique were crucial in consideration of bacteria as the tools for genetiC research. Significance of E. coli as popular organism for genetiC experimentation is due to (1) (2) (3) (4) (5) (6) (7)

Non-pathogenic nature. Single chromosome. Single copy of any gene. Short generation time (- 30 minutes). Ease in selection of mutants. Further studies confirmed natural phenomena of genetiC exchange-recombination existing in E. coli. Grows on simple nutrient media.

The advantages of E. coli as a popular candidate for genetiC engineering work are given in module 2. Here, we are still discussing the days of shifts from classical genetiCS to modern genetics. Subsequently, phages which were otherwise not paid attention (because they were expected to be medically useful to combat bacterial infections which they of course could not) also were considered important. Bacteriophages provide simple, easy to study chromosomes. Significance of phages in genetic research is due to (1) (2) (3) (4)

Simpler in structure. Shorter life cycle. Quantitative study is possible. Mutants can be obtained and can be easily selected.

From Classical Genetics to Modem Genetics

(5) (6)

(7)

7

Phage chromosomes, like bacterial chromosomes are capable of genetic exchange (crossing over) when present in same bacterial cell. There is no differentiation into male and female phages, so genetic recombination is assured by simultaneous infection of a given cell by more than one genetically distinct phage. Viruses have also provided useful models to study their interactions with hosts and expression of various genes.

000

Module 2

GENETIC ENGINEERING AND r-DNA TECHNOLOGY 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.1

What is genetic engineering? What is recombinant DNA technology? Common steps of recombinant DNA technology. Control of gene expression in prokaryotes and eukaryotes. Genetic engineering of E. coli. Genetic engineering of prokaryotes other than E. coli. Genetic engineering of Saccharomyces cerevisiae. Genetic engineering of fungi. Genetic engineering of mammalian cells. What is Genetic Engineering?

If there is any discipline which can claim a major share in the development and success of biotechnolog.y, it is genetics. Better health, ample food, environment control (clean environment), cheaper utility chemicals and energy, for all this and more; genetics has played a great role in biotechnology's achievements. Since biotechnology means 'applications of scientific and engineering principles to biological processes to provide goods and services', the control and better exploitation of biological processes would be possible if we have better control on the elements of heredity which govern them. Full understanding of biological processes is possible with detailed analysis of gene structure and function. Classical genetics has contributed to knowing of these basics and some efforts for improvement were also possible. We have really come a long way - structure of DNA - DNA as material for heredity - DNA replication - central dogma explaining passing of genetic information from DNA ~ RNA ~ Proteins synthesis - making mutants and studying their properties - natural processes of exchange of genetic information - mapping of chromosomes - operon model for gene functions - breeding techniques - limited improvements of useful (industrially) strains of micro organisms. And then in last 25 years, there has been tremendous progress in field of genetics. It has become a technology. The genetic manipulatiorl has reached to a new dimension. We call it genetic engineering. While differentiating from naturally occurring gene transfers or from conventional

(8)

Genetic Engineering and r-DNA Technology

9

breeding techniques, genetic engineering essentially involves some process in vitro. According to one definition, 'Genetic engineering' means the introduction of manipulated genetic material into a cell in such a way as to replicate and be passed on to progeny cells. Genetic engineering mayor may not, have recombinant DNA preparation step and with advancement of gene transfer technology, artificially DNA can be transferred to different host (host cells) without any vector and host organism can be engineered to carry desired properties. Genetic engineering includes(i) Gene isolation. (ii) DNA preparation. (iii) Preparing a vector-recombinant DNA. (iv) Introduction into host cell and stable maintenance. (v) Selection of transformed (transgenic) cells. (vi) Characterization and use of engineered organism. Thus, Genetic engineering implies (i) Techniques of engineering genes - r-DNA technology is part of this area. (ii) Strategies of engineering organisms. (iii) Applications of engineered organisms. Limitations of older techniques (mutations, breeding, etc.) are: (i)

(ii) (iii)

(iv) (v)

Time consuming. Changes in genome unpredictable. No reproducibility. More efforts required (e.g., lot of back-crossing to eliminate unwanted tra!ts and to get homozygous desired traits in breeding programs). No inter-specific transfer possible. (Taxonomic boundaries are not crossed in gene transfer)

Newer techniques of gene manipulation (genetic engineering which includes r-DNA technology) in comparison are (i)

Cheap. (ii) Convenient. (iii) Reproducible. (iv) More accurate. (v) Less time consuming. (vi) Inter-specific gene transfer possible. (Taxonomic boundaries are crossed in gene transfer) Many a time in the discussion of this topic the distinction is lost between the terms - genetic engineering and recombinant DNA technology. Today, if we are able to transfer and express a gene from plants or animals to bacteria and vice versa, this is possible because genetic code is assumed to be universal.

2.2

What is Recombinant DNA Technology? Recombinant DNA technology has revolutionised many fields. Recombinant DNA technology has added new weapons to the armoury of geneticist. Recombinant DNA technology has given new dimension to study and exploitation of cells. Recombinant DNA technology has taken manipulations to the gene level and further. Recombinant DNA technology has provided new approaches to the old problems. Recombinant DNA technology has helped to understand processes of mitosis, division, differentiation and development of whole animal better. Recombinant DNA technology helps in studies of biochemistry, to understand effects of various

10

Gene Biotechnology

proteins on cellular processes by under or overproducing these proteins. Recombinant DNA technology can be useful to many fields to get hard to purify proteins in large quantities. Recombinant DNA technology has important applications to gene mapping, detection of inherited diseases, cancer research, immunology, enzymology, industrial prodyctions. Thus, recombinant DNA technology which is the first part of genetic engineering enables us to achieve so much. Gene transfer is the key step (technique) in genetic engineering. The biggest advantage of genetic engineering as mentioned earlier is inter-specific transfer of genes. And one may question as to 'why do it?' The reasons are clear when we look at the innovative applications of it. Genetic engineering has enabled us to cross the taxonomic boundaries and now we can transfer, maintain and express gene from a source into unrelated species. The outcome is attractive, promising and that makes genetic engineering and biotechnology most sought after and hope-worthy. Some examples to stress the point are: (1) (2) (3) (4) (5) (6) (7) (8)

2.3

Insulin and other hormones which can be only available from higher animals in small quantities can be produced cheaply and amply in microorganism. Proteins of pharmaceutical value can be produced by animal tissue culturing or by animals themselves in their milk. Antiviral proteins like interferon can be produced cheaply and amply. Vaccines which are more potent and safe are produced and otherwise difficult to produce vaccines are produced in easily cultivable organisms. Pharmaceuticals can be produced by plant cells grown in suspension. Gene therapy is likely to be successful for genetic disorders. Quality, yields of plants and desirable traits like disease resistance, pesticide resistance can be introduced easily and with reproducibility. Nitrogen fixation by plants which may be a fantasy of yesterday is target of the research.

Common Steps of Recombinant DNA Technology Prepare DNA vector

.

J,

Cut DNA and modify

Isolate desired gene

J,

Modify as per requiryment

l-----7) Join DNA( J,

Isolate vector-gene combination e.g. by electrophoresis

J,

Introduce into host

J,

Select transformed or transgenic cells

J,

I" Characterise transgenic (engineered) Use the expression for study purpose ...----II organisms Only produce DNA sequence of interest in large quantity ....,J J, Cultivate engineered organisms on required scale

J,

Isolate and market the product

Recombinant DNA technology can have impact on production of fermentation products by following means:

11

Genetic Engineering and r-DNA Technology

(1)

It may cause increase in gene dosage of biosynthetic genes.

(2)

It may improve promoter activity.

(3)

It may eliminate feedback control of synthesis.

(4)

It may modify bio-synthetic genes to higher activity: (a)

by increasing turnover number, or

(b)

by improving stability.

(5)

It may modify organisms to use cheaper growth subtrates.

(6)

It may modify physiology to improve fermentation, e.g., reduced viscosity. r.-RNApol

L- ••p .• I

·1

0

I

lac y ~zomot~i I lac z lac A ~I-·-·-··~~--~I~I----~----~--------~I

lac I I

control sites

regulatory gene

structural genes

...I

L-mRNA 1 ________ 1 Repressor If binds to operator protein

+-

If Inducer added

structural genes transcription

~ prevents transcription

.

,----mRNA

of structural genes.

a---..

No "rotein product. can not bind operator

Repressor protein inactivated.

Fig. 2.1 Mechanism of Gene Expression in Prokaryotes. Example of lac Operon.

r_--.J

Structural gene

Non coding Enhancer

A

Promoter

....- - - - - -....

'\

Intron

flo .......

....... .

DNA template

"

RNA pol

CAP

~

Precursor mRNA

splicing

Nuclease + poly A polymerase

Ribosome

R

0

AAAAAAAAAA Mature m RNA Translation Protein product.

Fig. 2.2 Mechanism of Gene Expression In Eukaryotes.

12

2.4

Gene Biotechnology Control of Gene Expression in Prokaryotes and Eukaryotes

Although gene action is same and protein synthesis follows same pattern, mechanism of expression differs in prokaryotes and eukaryotic organisms. Also controls and regulatory mechanisms differ. Gene controls in bacteria are as explained in operon model (originally proposed by Jacob and Monod in 1961). Negative control, positive control and attenuation are the three mechanisms of control of gene action. These controls are primarily based on DNA-protein interactions. In negative control, repressor protein interacts with operator to inhibit transcription. In positive control, inducer binds to protein that stimulates transcription. And in attenuation, incomplete transcription operates, on genes responsible for amino acid synthesis. Particular amino acids availability decides continuation or termination of transcription process. Gene controls in plants and animals, are relatively less known, less understood. Selective gene expression during cell differentiation is a mystery. DNA in eukaryotes is as complex with histone and other proteins to form a structure known as chromatin. Significant alterations occur in chromatin structure during transcription and these changes will be associated with gene control mechanisms. Differences in gene control are: In Eukaryotes (i)

(ii) (iii) (iv) (v) (vi)

No direct equivalent of operon model. No polycistronic m-RNA. Each structural gene is transcribed separately with its own promoter, initiator, terminator. RNA polymerases are more complex. Non-coding sequences - enhancer elements present attract RNA polymerases to coding regions. RNA processing involves several steps (a) capping by 7-methyl guanosine to terminate transcription. (b) trimming of RNA with ribonucleases. (c) addition of poly(A) tail (20-250 nucleotides). (d) removal of introns by spliCing enzyme and ligating the coding sequences (exons). (e) post transcriptional modification - peptide cleavage, addition of prosthetic group, glycosylation, etc.

Genetic engineering may be carried out with variety of hosts (host cells) to carry the recombinant DNA. Requirements of genetic engineering, objectives of genetic engineering decide as to which host to use.

2.5

Genetic Engineering of E. coli

E. coli is the most popular choice and genes from various sources including those from other bacteria, yeasts, fungi, animals and plants have been successfully engineered in E. coli. Following are the reasons for using E. coli as host to express foreign genes. Advantages (1) E. coli is simple to grow on large scale.

(2) (3) (4)

Higher biomass productivities can be obtained. First restriction enzyme studied was from E. coli so it became an obvious choice in the beginning. Genetics and physiology of E. coli is amply known.

Genetic Engineering and r-DNA Technology (5) (6) (7)

(8) (9)

13

E. coli can serve as model molecular system for gene expression studies. Promoters, gene regulators are well understood. Suitable mutant strain of E. coli can provide an obvious host for gene bank made from another organism. E. coli and its phages are most easy to manipulate. Even simple calcium chloride treatment allows transformation of E. coli with plasmid. Abundance of well characterised promoters - like lac, tac, top and lactamose properties.

Disadvantages (1) E. coli do not usually get export of proteins into growth medium. Product is retained (2)

(3) (4)

(5) (6) (7) (8) (9)

2.6

within cells. Overexpressed foreign proteins form aggregates (inclusions) of denatured proteins. These are biologically non-active and need to be solubilised and refolded to get active protein. (However, advantage is their resistance to proteolysis.) E. coli not considered safe for r-DNA technology because it occurs naturally in the gut of human being and risks are not predictable. Regulatory agencies are not in favour. Endotoxin from E. coli often contaminates the proteins produced and it is difficult to remove it. E. coli cannot carry out post-translational modification for genes from mammals. Biological activity of the product is inadequate (not satisfactory). Imperfect spatial configuration leading to recognition of protein produced as foreign by targeted user organism. Damage to products by intra- and extracellular proteins. Problems of Plasmid stability, phage contamination also exist.

Genetic Engineering of Prokaryotes Other than E. coli Bacillus species and Streptomycetes are amongst the common prokaryotes other than E. coli

which are used for genetic engineering. Following are the reasons which are in favour of them -

Advantages (1) (2)

(3) (4) (5)

(6)

Industrially important organisms are not E. coli, but are Coryneforms, Streptomycetes, Bacilli, etc. Bacillus subtilils is naturally competent to take up DNA molecules, so it is technically advantageous. Bacillus species are particularly useful for enzymes production. Vegetative cell - spore stages enables us to study the effects of simplest forms of differentiation process on expression of foreign gene. Organisms other than E. coli are safer since they are not naturally present with human. Degradations, antibiotic production, enzyme production, other production, plant infections are not part of E. coli. Therefore, expression and application is not much successful in E. coli while other prokaryotes should be more suitable. Non-pathogenic, easy to culture and release of proteins in growth medium are advantages. Product recovery is easy and downstream processing is less expensive.

Disadvantages (1) (2)

Insufficient knowledge of gene regulation mechanisms. Plasmid instability is one of the problems associated with Bacillus.

14

Gene Biotechnology

(3) (4)

2.7

Level of expression of foreign genes is not high. Proteases produced by Bacillus can be harmful to heterologous proteins produced by Bacillus.

Genetic Engineering of Saccharomyces cerevislae: Saccharomyces cerevisiae is normally the next choice after E. coli:

Advantages (Reasons) (1) Growth requirements are simple. (2) Large scale growth of yeasts is possible. (3) Ability to grow in wide variety of environments. (4) High cell densities can be reached. (5) Saccharomyces cerevisiae is industrially important in bakery, brewery, etc. (6) Saccharomyces cerevisiae is a eukaryote and yet possesses plasm ids. Being eukaryote it is likely to express mammalian proteins better than in E. coli. (7) Genetics and physiology of Saccharomyces cerevisiae is adequately known. (8) S. cerevisiae is one of the few eukaryotes where genes can be specifically targeted to chromosomal locations and chromosomal genes can be replaced or mutated by gene transfer techniques. (9) High level expression systems are developed in S. cerevisiae. Cost effective, versatile and easy production. (10) Yeasts do not produce pyrogens or endotoxins and hence can be used for expression and production of mammalian proteins. (11) Yeasts are not considered as pathogens, so are acceptable by regulatory agencies. Disadvantages (1) Although post-transnational modification occurs in S. cerevisiae, it is not same as in animal cells. (2) Heterologous proteins when produced can form inclusions (aggregates). (3) Much is still to be learnt about gene regulation. 2.8

Gienetlcs Engineering of Fungi

Fungi due to their structure, life cycle, taxonomic position and also industrial importance provide special problems and opportunities for genetic engineering. Fungi are industrially useful as producers of antibiotics, enzymes, vitamins. Fungi are causing plant and animal diseases. Fungi have a role as decomposers and as mycorrhizae. Genetic engineering of fungi can have various applications. Following are some of the features in favour and against genetiC engineering of fungi. Promoters, regulatory elements, secretion and processing signals of fungal gene (5' region) are used for expression of heterologous protein.

Advantages (1) Range of organisms available are simple unicellular to well differentiated multicellular. (2) Fungi are relatively simple eukaryotes. (3) Fungi sporulate profusely and yet have vegetative mycelium stage. (4) Fungi can be grown on large-scale in simple media like other microorganisms. (5) Fungi have large surface area to volume ratio which favours protein export.

Genetic Engineering and r-DNA Technology

(6)

15

Due to relative simplicity and yet multistage nature, fungi can be used as model for more complex developmental pathways in mammals.

Disadvantages (1) Gene regulation mechanisms are poorly understood. (2) Susceptibility of heterologous proteins to fungal (secreted) protease enzymes. (3) Good expression systems are lacking. (4) Difficulty of obtaining high levels of secreted mammalian proteins may relate to fundamental differences in the secretion pathways of fungi and animals. 2.9

Genetic Engineering of Mammalian Cells

Either animal cells in culture or whole animals are the targets of genetic engineering. Transgenic animals can be produced with the idea of using them as bioreactors to produce pharmaceutical proteins in milk. Transgenic mouse is produced with the idea of using it as a model for study of expression of foreign gene in higher mammals. Somatic cell engineering in mammals is legally and ethically accepted. ~ene therapy is aimed at to fight with various genetic disorders. Animal cells in culture are genetically engineered to get production of pharmaceutical proteins, vaccines which otherwise is difficult.

Advantages (1) Less impurities while producing pharmaceutically important proteins. (2) Proteins are released in growth medium. (3) Desired post-transnational modification occurs and proteins produced are likely to be less immunogenic to humans. (4) Good expression systems are available.

~

Disadvantages (1) Mammalian cells in culture are delicate to handle. (2) Mammalian cell culturing is costly when large scale growth is desired. (3) Mammalian cells are slow growing and require defined media with many supplements. (4) Mammalian cells in culture are subject to disruption by shear forces. (5) Great care has to be taken to avoid contamination.

000

Module 3

GENETIC COMPLEXITY 3.1 3.2 3.3 3.4 3.5 3.6

The prokaryotic genome. The eukaryotic genome. Promoters. Enhancers. Transcription initiation signals. Reverse genetics.

3.1

The Prokaryotic Genome E. coli has been the prokaryotic organism in which nuclear organization has been most extensively investigated. However, other prokaryotic organisms are also expected to share this distinctive nuclear organization. The bacterial nucleus may be defined as the region of the cell in which single molecule of DNA representing bacterial chromosome is packed. Bacterial chromosome is highly charged molecule due to phosphates associated with base pairs and hydroxyl ions. Bacterial chromosome (DNA) is not coupled with any basic proteins, or histones as in eukaryotes. Negative charge of bacterial DNA is neutralised by small amounts of polyamines (spermine and spermidine). Bacterial DNA has 1.36 mm contour length and thickness of 2 nm. E. coli DNA contains around 4 million nucleotides with 4000 genes. It is a supercoiled structure with 50 or more loops and is tightly and compactly packed in nuclear zone held together with some RNA which forms core of 9 condensed chromosome. Molecular weight of double stranded E. coli - DNA is 2.8 x 10 • Bacterial chromosome (DNA) has a single copy of each of the gene that it possesses. In prokaryotes many genes are transcribed as single units (monocistronic mRNA). However, there are also some genes which form clusters. They encode enzymes that are part of the same metabolic pathway. These clusters of genes are called operons and are usually under the control of one promoter sequence from which a single RNA transcript (polycistronic mRNA) that has the information for several different proteins is produced. The initiation of transcription begins at a specific nucleotide in one of the two DNA strands. First nucleotide of the transcribed RNA molecule is designated as the + 1 nucleotide. Nucleotides further in the template strand are aSSigned (+) numbers and are said to be downstream while those nucleotides that precede the +1 nucleotide are said to be upstream and are assigned (-) numbers. Thus, -10 site is Pribnow box. (binding site for RNA polymerase enzymes). (16)