Gene cloning involves copying a gene and inserting it into a self-replicating vector to produce multiple copies of the gene. PCR (polymerase chain reaction) is a technique used to amplify specific genes. Both gene cloning and PCR are important for obtaining pure samples of genes and amplifying them for various applications like sequencing, expression of proteins, and genetic engineering.

1. Gene cloning
By-PRIYA TAMANG

2. Introduction
• Gene cloning is a common practice in molecular biology labs
• It is used to create copies of a particular gene for downstream applications,
such as sequencing, mutagenesis, genotyping or heterologous expression of
a protein.
• The traditional technique for gene cloning involves the transfer of a DNA
fragment of interest from one organism to a self-replicating genetic
element, such as a bacterial plasmid.
• This technique is commonly used today for isolating long or unstudied
genes and protein expression.
• A more recent technique is the use of polymerase chain reaction (PCR) for
amplifying a gene of interest.
• The advantage of using PCR over traditional gene cloning, is the decreased
time needed for generating a pure sample of the gene of interest.
• However, gene isolation by PCR can only amplify genes with
predetermined sequences. For this reason, many unstudied genes require
initial gene cloning and sequencing before PCR can be performed for
further analysis.

3. • A clone is an exact copy of an organism, organ, single
cell, organelle or macromolecule.
• Cell lines for medical research are derived from a single
cell allowed to replicate millions of times, producing
masses of identical clones.
• Gene cloning is the act of making copies of a single gene.
• Cloning can provide a pure sample of an individual
gene, separated from all the other genes that it normally
shares the cell with.
• Once a gene is identified, clones can be used in many
areas of biomedical and industrial research.
• Genetic engineering is the process of cloning genes into
new organisms, or altering a genetic sequence to change
the protein product.
What is gene cloning?

4. History
• In 1922, morghan and his colleagues developed the technique for gene
mapping.
• In 1903,W.sutton proposed the idea of a gene residue on chromosome.
• By 1922, they had analysis the relative positions of over 2000 genes on
the 4th chromosomes of fruit fly, drosophila melongata.
• Until 1940’s, there was no real understanding of molecular nature of
gene.
• In 1944, the experiments of Avery, McLeod, and McCarty and in 1952,
horshey and chase, stated that DNA ( deoxyribose nucleic acid) is the
genetic material: up until then it was thought that genes were made up
of protein.
• Discovery in the role of DNA was tremendous stimulus to the genetic
research.
• Delbruck, chargeff, crick and monad, contributed in the second great
age of genetics.
• Between 1952 and 1966, in this years the structure of DNA was
elucidated, the genetic code cracked, and the process of transcription
and translation.

5. • There was a period of anticlimax, some molecular biologist was in state
of frustration that the experimental techniques of the late 1960’s were
not sophisticated enough to allow the gene to studied in any greater
extends.
• During 1971-73, there was a revolution thrown back into gear by
introducing completely new methodology, recombinant-DNA
technology or genetic engineering.
• This new methodology as heir core in the process of gene cloning, it
sparkled as another great age of genetics.
• It led to rapid and efficient DNA sequencing techniques that enabled
the structure of individual genes to be determined, reaching
culmination with the massive genome sequencing projects including the
Human genome project which was completed in 2000.
• In 1985, Kary Mullis invented the PCR , an exquisitely simple technique
that acts as a perfect complement to Gene Cloning.
• PCR has made easier many of the technique, that were possible but
difficult to carry out when gene cloning was used on it own.
• It extended to the range of DNA analysis and enabled molecular biology
to find range in the field of medicine, agriculture, and biotechnology.
• With the invention of PCR , the Archeogenetics, molecular biology, and
DNA Forensics have to become possible.
• 40 years passed since the dawning of age of gene cloning, but there is no
end to the excitement in sight.

6. Fundamental steps
• Identification and isolation of the desired gene or DNA
fragment to be cloned.
• Insertion of the isolated gene in a suitable vector.
• Introduction of this vector into a suitable organism/cell called
host.
• The vector multiplies within the host cell, producing
numerous identical copies not only of itself but also of the
gene that it carries.
• During the division of the host cell, copies of the recombinant
DNA molecules are passed to the progeny and further vector
replication takes place.
• After a large number of cell divisions, a colony, or clone, of
identical host cells is produced. Each cell in the clone contains
one or more copies of the recombinant DNA molecule.

8. What is PCR?
• PCR is a method of copying DNA molecules. DNA replication is common in
life; for example it takes place inside your own cells every time they divide.
An enzyme known as polymerase uses one strand of DNA as a template to
create a complementary strand. The result is that one double stranded DNA
molecule is converted into two, both identical to the first.
• PCR, or the polymerase chain reaction, adds two components to this
process. The initial reaction yields twice the number of starting molecules,
but then is immediately followed by a subsequent reaction, which yields
twice the molecules as the first reaction. This is why PCR is known as a
chain reaction. Commonly 25-40 reactions are chained together,
theoretically resulting in 225 – 240 more molecules of DNA then were
initially present.
• Additionally, the goal of a PCR reaction is commonly to replicate only a
portion of the genome of interest. For example, somewhere between 75-
1000 bases, instead of the entire human genome of 3 billion bases. As PCR
produces billions of copies of only the DNA of interest, this process is
known as “amplification”.

9. Why is PCR important?
• The amplification provided by PCR is very powerful. For
example, suppose we want to detect whether a dangerous E.
Coli pathogen is present in a sample of meat. That meat
sample contains a huge amount of DNA from the meat source,
and many non-pathogenic bacteria. Looking for the DNA
from the pathogenic E. Coli, is akin to searching for a needle
in a haystack.
• However a PCR reaction can be designed to amplify only the
DNA from a portion of this pathogenic E. Coli. If the pathogen
is present, we can make billions of copies of its targeted DNA,
which will come to outnumber the overall DNA originally
present in the sample, and allow us to easily detect it. If no
such signal is amplified by a properly controlled reaction, we
can conclude the pathogen was not present.

10. How is it used?
• PCR and related techniques have many applications.
Here are just a few
• Human Diagnostics
▫ Detecting viral infections (HIV, etc.)
▫ Detecting bacterial infections (Tuberculosis, etc.)
▫ Genotyping (detecting genetic variants, which can indicate
predisposition to disease)
• Environmental Monitoring
▫ Water quality monitoring
▫ Food safety testing
• Scientific Research
▫ Preparing DNA to sequence
▫ Monitoring gene expression levels
▫ Manipulating DNA in genetic engineering and synthetic
biology

11. How does PCR work?
• The principles behind every PCR, whatever the sample of DNA, are
the same.
• Five core ‘ingredients’ are required to set up a PCR. We will explain
exactly what each of these do as we go along. These are:
▫ the DNA template to be copied
▫ primers, short stretches of DNA that initiate the PCR reaction, designed
to bind to either side of the section of DNA you want to copy
▫ DNA nucleotide bases? (also known as dNTPs). DNA bases (A, C, G and
T) are the building blocks of DNA and are needed to construct the new
strand of DNA
▫ Taq polymerase enzyme? to add in the new DNA bases
▫ buffer to ensure the right conditions for the reaction.
• PCR involves a process of heating and cooling called thermal cycling
which is carried out by machine.

12. There are three main stages:
▫ Denaturing – when the double-stranded template DNA is heated to
separate it into two single strands.
▫ Annealing – when the temperature is lowered to enable the DNA
primers to attach to the template DNA.
▫ Extending – when the temperature is raised and the new strand of
DNA is made by the Taq polymerase enzyme.

13. • These three stages are
repeated 20-40 times,
doubling the number of
DNA copies each time.
• A complete PCR
reaction can be
performed in a few
hours, or even less than
an hour with certain
high-speed machines.
• After PCR has been
completed, a method
called electrophoresis
can be used to check the
quantity and size of the
DNA fragments
produced.

14. What happens at each stage of PCR?
•Denaturing stage
▫ During this stage the cocktail containing the template
DNA and all the other core ingredients is heated to 94-
95⁰C.
▫ The high temperature causes the hydrogen
bonds? between the bases in two strands of template
DNA to break and the two strands to separate.
▫ This results in two single strands of DNA, which will
act as templates for the production of the new strands
of DNA.
▫ It is important that the temperature is maintained at
this stage for long enough to ensure that the DNA
strands have separated completely.
▫ This usually takes between 15-30 seconds.

15. • Annealing stage
▫ During this stage the reaction is cooled to 50-65⁰C. This enables
the primers to attach to a specific location on the single-stranded
template DNA by way of hydrogen bonding (the exact
temperature depends on the melting temperature of the primers
you are using).
▫ Primers are single strands of DNA or RNA? sequence that are
around 20 to 30 bases in length.
▫ The primers are designed to be complementary? in sequence to
short sections of DNA on each end of the sequence to be copied.
▫ Primers serve as the starting point for DNA synthesis. The
polymerase enzyme can only add DNA bases to a double strand of
DNA. Only once the primer has bound can the polymerase
enzyme attach and start making the new complementary strand
of DNA from the loose DNA bases.
▫ The two separated strands of DNA are complementary and run in
opposite directions (from one end - the 5’ end – to the other - the
3’ end); as a result, there are two primers – a forward primer and
a reverse primer.
▫ This step usually takes about 10-30 seconds.

16. • Extending stage
▫ During this final step, the heat is increased to 72⁰C
to enable the new DNA to be made by a special Taq
DNA polymerase enzyme which adds DNA bases.
 Taq DNA polymerase is an enzyme taken from the
heat-loving bacteria Thermus aquaticus.
 This bacteria normally lives in hot springs so can
tolerate temperatures above 80⁰C.
 The bacteria's DNA polymerase is very stable at high
temperatures, which means it can withstand the
temperatures needed to break the strands of DNA
apart in the denaturing stage of PCR.
 DNA polymerase from most other organisms would
not be able to withstand these high temperatures, for
example, human polymerase works ideally at 37˚C
(body temperature).

17. • 72⁰C is the optimum temperature for the Taq
polymerase to build the complementary strand. It
attaches to the primer and then adds DNA bases to the
single strand one-by-one in the 5’ to 3’ direction.
▫ The result is a brand new strand of DNA and a double-
stranded molecule of DNA.
• The duration of this step depends on the length of DNA
sequence being amplified but usually takes around one
minute to copy 1,000 DNA bases (1Kb).
• These three processes of thermal cycling are repeated
20-40 times to produce lots of copies of the DNA
sequence of interest.
• The new fragments of DNA that are made during PCR
also serve as templates to which the DNA polymerase
enzyme can attach and start making DNA.
• The result is a huge number of copies of the specific DNA
segment produced in a relatively short period of time.

18. Why gene cloning and PCR are so
important?

19. • Obtaining a
pure sample of
a gene by
cloning

20. The
problem
of
selection.

21. PCR can also be used to purify a gene
Gene isolation by PCR

22. Cloning applications
• Gene cloning has made a phenomenal impact on the speed of
biological research and it is increasing its presence in several areas
of everyday life. One of the reasons why biotechnology has received
so much attention during the last decade is because of gene cloning.
• Production of recombinant protein
▫ Proteins that are normally produced in very small amounts include
growth hormone, insulin in diabetes, interferon in some immune
disorders and blood clotting factor VIII in hemophilia, are known to be
missing or defective in various disorders. Prior to the advent of gene
cloning and protein production via recombinant DNA techniques, these
molecules were purified from animal tissues or donated human blood.
But both sources have drawbacks, including slight functional differences
in the non human proteins and possible viral contamination. (e.g. HIV,
CJD). Production of protein from a cloned gene in a defined, non
pathogenic organism would circumvent these problems. A gene for an
important animal or plant protein can be taken from its normal host,
inserted into a cloning vector, and introduced into a bacterium. If the
manipulations are performed correctly then the gene will be expressed
and the protein is synthesized by the bacterial cell. Then it is possible to
obtain large amounts of the protein.
But in practice obtaining recombinant protein is not as easy as
theoretically it sounds. For this special types of cloning vectors are
needed.

24. Expression of foreign genes in E.coli
• Expression of a foreign gene in E.coli is dependent on the collection
of signals surrounding the gene. These signals, which are short
sequences of nucleotides, advertise the presence of the gene and
provide instructions for the transcriptional and translational
apparatus of the cell. The three most important signals
for E.coli genes are
▫ 1) the promoter, at which transcription should start,
▫ 2) the terminator,at which transcription should stop, and
▫ 3) the ribosome binding site, a short nucleotide sequence
recognised by the ribosome as the point at which it should attach
to the mRNA molecule.
• The foreign gene is inserted into a vector in such a way that the gene
is placed under control of a set of E.coli expression signals. Cloning
vehicles which provide these signals, and which can therefore be
used in the production of recombinant protein, are
called expression vectors.

25. • An efficient expression requires a strong promoter,
an E.coli ribosome binding sequence and a terminator.
• As the foreign gene is inserted into a unique restriction site
present in the middle of the expression signal cluster, so a
cassette is formed by these expression signals in most of the
vectors.
• Ligation of the foreign gene into the cassette therefore places
it in the ideal position relative to the expression signals.
• Insertion of the foreign gene into this restriction site must be
performed in such a way as to fuse the two reading frames,
producing a hybrid gene that starts with the E.coli segment
and progresses without a break into the codons of the foreign
gene.
• The product of gene expression is therefore a hybrid protein,
consisting of the short peptide coded by the E.coli reading
frame fused to the amino-terminus of the foreign protein.

27. Problems with the production of
recombinant protein in E.coli
• The problems associated with the production of protein from foreign genes cloned in E.coli can be
grouped into two categories: those that are due to the sequence of the foreign gene, and those that
are due to the limitations of E.coli as a host for recombinant protein synthesis.
• The problems associated with the sequence of the foreign gene are:
• 1) The foreign gene might contain introns, this would be a major problem as E. coli genes don't
contain introns and the bacterium therefore doesn't posses the necessary machinery for removing
introns from transcripts.
• 2) The foreign gene might contain sequences that act as termination signals in E.coli.
• 3) The codon usage of the gene may not be ideal for translation in E.coli. These problems can
usually be solved, though the necessary manipulations may be time consuming and costly.
• The problems associated with E.coli are:
• 1) E.coli might not process the recombinant protein correctly.
• 2) E.coli might not fold the recombinant protein correctly. If the protein doesn't take up its
correctly folded, tertiary structure then usually it is insoluble and forms an inclusion body within
the bacterium. Its nearly impossible to convert the protein into it's correctly folded form. The
protein is inactive under these circumstances.
• 3) E.coli might degrade the recombinant protein.
•
The problems associated with obtaining high yields of active recombinant proteins from genes
cloned in E.coli have led to the development of expression system for higher organisms. Yeasts and
fungi can be grown just as easily as bacteria in continuous culture, and may express a cloned gene
from a higher organism.

28. Recombinant protein from yeast
• The yeast Saccharomyces cerevisiae is the most popular
microbial eukaryote for recombinant protein production.
Cloned genes are often placed under the control of the GAL
promoter, which is normally upstream of the gene coding for
galactose epimerase, enzyme involved in the metabolism of
galactose. The GAL promoter is induced by galactose,
regulating expression of a cloned gene.
• Yields of recombinant proteins are relatively high,
but S.cerevisiae is unable to glycosylate animal proteins
correctly and lacks an efficient system for secreting proteins
into the growth medium. In the absence of secretion,
recombinant proteins are retained in the cell and are
consequently less easy to purify. Besides these
drawbacks, S.cerevisiae is the most frequently used microbial
eukaryote for recombinant protein synthesis.

29. Recombinant protein from
filamentous fungi
• Advantages of fungi in recombinant protein production
lie in their good glycosylation properties and their ability
to secrete proteins into the growth medium. The wood
rot fungus Trichoderma reesei,in its natural habitat
secretes cellulolytic enzymes that degrade the wood that
it lives on. These fungi produce recombinant proteins in
a form that aids purification. The fungus Aspergillus
nidulans is popular for producing recombinant proteins.
Expression vectors for A.nidulans usually carry the
glucoamylase promoter, induced by starch and repressed
by xylose.

30. Animal cells in recombinant protein
production
• For proteins with complex and essential glycosylation
structures an animal cell might be the only type of host
within which the active protein can be synthesized. A
problem with some animal cell lines is that they require
a solid surface on which to grow, adding complications
to the design of culture vessels. Rates of growth and
maximum cell densities are much for animal cells
compared with microorganisms, limiting the yield of
recombinant protein. Two promoters that have been
used in mammalian cells are the heat shock promoter of
the human hsp-70 gene, which is induced at
temperatures above 40°C, and the mouse
metallothionein gene promoter, which is switched on by
addition of zinc salts to the culture medium.

31. Gene cloning in medicine
• Medicine will continue to be a major beneficiary of the
gene cloning revolution.
Production of recombinant insulin
• Insulin, synthesized by the β-cells of the Islets of Langerhans in the pancreas, controls the
level of glucose in the blood. Insulin deficiency will result in the disease diabetes mellitus,
which is a life threatening disease if left untreated. Insulin is a relatively small protein
molecule, comprising two polypeptides. In humans these are synthesized as a precursor
called preproinsulin which contains the A and b segments linked by a third chain C and
preceded by a leader sequence. The leader sequence is removed after translation and the C
chain is excised, leaving the A and B polypeptides linked to each other by two disulphide
bonds.
• For synthesis and expression of artificial insulin genes, two recombinant plasmids were
constructed, one carrying the artificial gene for the A chain and one the gene for the B
chain. In each case, the artificial gene was ligated to a lacZ' reading frame present in a
pBR322-type vector. The insulin genes were therefore under the control of the strong lac
promoter and were expressed as fusion proteins, consisting of the first few amino acids of
β-galactosidase followed by the A or B polypeptides. Each gene was designed so that its β-
galactoatsidase and insulin segments were separated by a methionine residue, so that the
insulin polypeptides could be cleaved from the β-galactosidase segments by treatment with
cyanogen bromide. The purified A and B chains were then attached to each other by
disulphide bond formation in the test tube. A subsequent improvement has been to
synthesize not the individual A and B genes, but the entire proinsulin reading frame,
specifying B chain-C chain- A chain. The pro-hormone can fold spontaneously into the
correct, disulphide- bonded structure. The C chain segment can then be excised relatively
easily by proteolytic cleavage.

32. Synthesis of other recombinant human proteins and
recombinant vaccines
• Many growth factors are have been synthesized from gene cloned in
bacteria and eukaryotic cells. These growth factors include somatostatin,
somatotropin, factor VIII, Interferon-α, Interferon-β, Interferon-γ,
interleukins etc. which are used in tratment of various diseases and also
some of them having potential uses in cancer therapy. These proteins are
synhesized in very limited amounts in the body, so recombinant technology
is the only viable means of obtaining them in the quantities needed for
clinical purposes.
•
Production of vaccines as recombinant proteins
• The use of gene cloning in this field centres on the discovery that virus specific antibodies
are sometimes synthesized in response to not only to the whole virus particle, but also to
isolated components of the virus. This is particularly true of purified preparations of the
proteins present in the virus coat. If the genes coding for the antigenic proteins of a
particular virus could be identified and inserted into an expression vector, then the
method for the synthesis of animal protein could be employed in the production of
recombinant proteins that might be used as vaccines. These vaccines would have the
advantages that they would be free of intact virus particles and they could be obtained in
large quantities. But this approach hasn't entirely been successful, because recombinant
coat proteins often lack the full antigenic properties of the intact virus. But one success has
been achieved with hepatitis B virus, whose coat protein has been synthesized in
Saccharomyces cerevisiae, using a vector based on 2μm plasmid. The protein was obtained
in reasonably high quantities and when injected into monkeys provided protection against
hepatitis B virus. This recombinant vaccine has been approved for use in humans.
• Live recombinant vaccine: Recombinant vaccinia viruses could be used as live vaccines
against other diseases. If a gene coding for a virus coat protein, is ligated into the vaccinia
genome, under control of a vaccinia promoter, then the gene will be expressed. After
injection into the blood stream, replication of the recombinant virus will result not only in
production of new vaccinia particles, but also in significant quantities of the major surface
antigen.

33. • Live recombinant vaccine
▫ Recombinant vaccinia viruses could be used as live vaccines
against other diseases. If a gene coding for a virus coat
protein, is ligated into the vaccinia genome, under control
of a vaccinia promoter, then the gene will be expressed.
After injection into the blood stream, replication of the
recombinant virus will result not only in production of new
vaccinia particles, but also in significant quantities of the
major surface antigen.
• Role of gene cloning in identification of genes
responsible for human diseases
▫ A genetic disease is caused by a defect in a specific
gene. Individuals carrying the defective gene are being
predisposed towards developing the disease at some stage
of their lives. Gene identification may provide an indication
of the biochemical basis to the disease, enabling therapies
to be designed. Identification of the mutation present in a
defective gene can be used to devise a screening programme
so that the mutant gene can be identified in individuals who
are carriers or who have not yet developed the disease.
Identification of the gene is a prerequisite for gene therapy.

34. • Mapping the breast cancer gene BRCA1
▫ Mapping of a gene accurately is required for gene isolation. Linkage analysis
involves comparing the inheritance pattern for the target gene with the
inheritance patterns for genetic loci whose map positions are already known. If
two loci are inherited together then they must be very close on the same
chromosome. With breast cancer, the first breakthrough occurred in 1990 as a
result of RFLP linkage analysis carried out by a group at the University of
California at Berkeley. This study showed that in families with a high incident of
breast cancer a significant number of women who suffered from the disease all
possessed the same version of an RFLP called D17S74.This RFLP had previously
been mapped to the long arm of chromosome 17. BRAC1 must therefore also be
located on the long arm of chromosome 17. For pinpointing the BRAC1 more
accurately, the chromosomal region containing BRAC1 was examined for tendem
repeat sequences, similar to those used in genetic fingerprinting, but with very
short repeat units. These repeat sequences are useful in linkage analysis as the
number of repeats at a locus is highly variable, probably because occasional errors
in DNA replication leads to additional units being inserted. Linkage is assessed by
comparing the inheritance patterns of a locus of a particular length and the gene
under investigation. This approach, using the repeat loci identified on
chromosome 17, reduced the size of BRAC1containing region from 20 Mb down to
just 600kb.
• Gene therapy
▫ Gene therapy is the final application of cloning in medicine. Gene therapy is the
insertion, alteration, or removal of genes within an individual's cells and
biological tissues to treat disease. It is a technique for correcting defective genes
that are responsible for disease development.

35. • Somatic cell therapy
• It involves manipulation of ordinary cells, usually ones which can be removed from the
organism, transfected and then placed back in the body. The technique has most promise
for inherited blood diseases, with genes being introduced into stem cells from the bone
marrow, which give rise to all the specialized cell types in the blood. Somatic cell therapy
also has potential in the treatment of lung diseases such as cystic fibrosis, as DNA
introduced into the respiratory tracts of rats via an inhaler is taken up by the epithelial cells
in the lungs, although gene expression occurs for only a few weeks.
•
Germline therapy
• In germline therapy, a fertilized egg is provided with a copy of the correct version of the
relavant gene and reimplanted into the mother. If successful, the gene is present and
expressed in all cells of the resulting individual. Germline therapy is usually carried out by
microinjection of DNA into the isolated egg cell and theoretically could be used to treat any
inherited disease.
•
• Before gene therapy can become a permanent cure for any condition, the therapeutic DNA
introduced into target cells must remain functional and the cells containing the therapeutic
DNA must be long-lived and stable. Problems with integrating therapeutic DNA into the
genome and the rapidly dividing nature of many cells prevent gene therapy from achieving
any long-term benefits. Patients will have to undergo multiple rounds of gene therapy.
•
• Gene cloning in agriculture
• Gene cloning provides a new dimension to crop breeding by enabling direct changes to be
made to the genotype of a plant, circumventing the random processes inherent in
conventional breeding. In gene addition, cloning is used to alter the characteristics of a
plant by providing it with one or more new genes. In gene subtraction, genetic
engineering techniques are used to inactivate one or more of the plant's existing genes.

36. Gene addition
• Plants are subject to predation by virtually all other types of organisms- viruses,
bacteria, fungi and animals but in agriculture, the greatest problems are caused
by insects. To reduce losses by insects, crops are regularly sprayed with
insecticides. Several of these insecticides have potentially harmful side effects on
the environment. An ideal insecticide should be toxic only to the insect targeted
and not to any other insects, animals and humans. The insecticide should also
be biodegradable, so that any residues remain after the crop is harvested, or
which are carried out of the field by rain water, don't persist long enough to
damage the environment.
• Insects along with plants occasionally use bacteria as their diet. Several
types of bacteria have evolved defence mechanisms against insect predation, for
example Bacillus thuringiensis during sporulation forms intracellular crystalline
bodies that contain an insecticidal protein called the δ-endotoxin which is highly
poisonous to insects. It is an inactive precursor and after ingestion by the insect
this pro-toxin is cleaved by proteinases, resulting in shorter versions of the
protein that display the toxic activity, binding to the inside of insect's gut and
damaging the surface epithellium so that the insect is unable to feed and
consequently starves to death. Biodegradability of B.thuringiensis acts as a
disadvantage so they must be reapplied at regular intervals during the growing
season which is not cost effective. The δ-endotoxins that do not require regular
application are developed by modifying the structure of toxin via protein
engineering to be more stable.

37. • Maize is a crop plant that is not served well by conventional insecticides. A
major pest of the plant is the European corn borer, which tunnels into the
plant from eggs laid on the under-surfaces of leaves, thereby evading the
effects of insecticides applied by spraying. The first attempt at countering
this pest by engineering maize plants to synthesize δ-endotoxin was made
by plant biotechnologists at a Ciba-Geigy laboratory in North Carolina.
They worked with the Cry1A(b) version of the toxin which had previously
been shown to be a 1155-amino-acid protein, with the toxic activity residing
in the segment from amino acids 29 to 607. Instead of isolating the natural
gene they made a shortened version of the first 648 codons, by artificial
gene synthesis. The artificial gene was ligated into a cassette vector between
a promoter and polyadenylation signal from cauliflower mosaic virus, and
introduced into maize embryos by bombardment with DNA coated
microprojectiles. The embryos were grown into mature plants, and
transformants identified by PCR analysis of DNA extracts, using primers
specific for a segment of the artificial gene.
• In the immunological tests the results showed that the artificial gene
was indeed active, but the amount of δ-endotoxin being produced varied
from plant to plant. This was probably due to positional effects, the level of
expression of a gene cloned in a plant often being influenced by the exact
location of the gene in the host chromosomes.

38. • Gene subtraction
▫ Gene subtraction is a modification which involves the inactivation
of a gene rather than its removal. The most successful so far in
practical terms is the use of antisense technology.
• Antisense technology
▫ In antisense technology the gene to be cloned is ligated into the
vector in reverse orientation.When this cloned gene is
transcribed, the RNA synthsized is the reverse complement of the
mRNA from the normal sequence. This antisense RNA is able to
prevent synthesis of the product of the gene it is directed against,
this is due to the hybridization between antisense and sense
copies. This block the expression by degrading double-stranded
RNA by cellular ribonucleases. It prevents ribosomal attachment
to sense strand.

39. Application
• The role of polygalacturonase gene in tomato
fruit ripening
▫ In tomato within six weeks after flowering, a number of genes involved
in the later stages of ripening are switched on, including one coding for
the polygalacturonic enzyme. This enzyme slowly breaks down the
polygalacturonic acid component of the cell walls in the fruit pericarp,
resulting in a gradual softening. Partial inactivation of the
polygalacturonase gene increases the time between flavour development
and spoilage of the fruit.
•
• Cloning the antisense polygalacturonase
gene
▫ A 730-bp restriction fragment was obtained from the 5' region of the
normal polygalactujronase gene. A plant polyadenylation signal was
attached to the beginning of this fragment, a cauliflower mosaic virus
promoter was ligated to the end, and the construction was inserted into
the Ti plasmid vector pBIN19. Inside the plant, it synthesizes antisense
RNA complementary to polygalacturonase mRNA.
▫ The recombinant pBIN19 molecules were introduced into
Agrobacterium tumefaciens bacteria and then the bacteria were allowed
to infect tomato stem segments. Small amount of callus material
collected from surface of these segments were tested for their ability to
grow on an agar medium containing kanamycin. Resistant transformants
were identified and allowed to develop into mature plants.