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L12 APPLICATIONS OF RECOMBINANT DNA TECHNIQUES

HOW CLONED CDNA ENCODING THE LIGHT AND HEAVY CHAINS FROM A
MOUSE MONOCLONAL ANTIBODY CAN BE EXPRESSED IN PLANTS.
 The mouse monoclonal antibodies are identical immunoglobulins that are all derived from
the same clone of plasma cells.
 The cDNAs encoding their light (L) and heavy (H) chains are ligated into separate T-DNA
vectors, and placed under the control of a constitutive CaMV promoter.
 The H and L chain cDNAa contain signal sequences from mouse antibody. The signal
sequence is the N-terminal sequence of a secreted protein, which is required for
transport through the cell membrane.
 The plasmids are transferred separately into tobacco plants by Agrobacterium infection.
 Transgenic plants containing the light- and heavy-chain genes are sexually crossed to
produce progeny plants that contain both genes and that express functional antibody.
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PRODUCTION OF RECOMBINANT GROWTH HORMONES IN E.COLI

At the same time when recombinant insulin was being made in E. coli, research on the
recombinant growth hormones, somatostanin and somatotropin was going on. These two proteins
act in conjunction to control growth processes in the human body. Their malfunction leads to
painful and disabling disorders such as uncontrolled bone growth (agromegaly) and dwarfism.

Somatostanin was the first human protein to be synthesized in E. coli. Being a very short
protein, only 14 amino acids in length, it was ideally suited for artificial gene synthesis. The
procedure for its synthesis was the same as for insulin. It involved insertion of the artificial gene
into a lacZ’ vector, synthesis of a fusion protein, and cleavage with cyanogen bromide.

Somatotropin is 191 amino acids in length, equivalent to almost 600 bp. It is produced in the
pituitary gland. Its deficiency in children results in hypopituitary dwarfs who never achieve
normal stature. Regular injection of somatotropin stimulates growth in such children to almost
normal heights. Before recombinant DNA technology, somatotropin was obtained from human
corpses, but a problem arose when a number of children were infected with a fatal virus in
somatotropin from one of the corpses. Production of recombinant somatotropin was safe, reliable
and plenty of the hormone could be obtained.

The strategy for its synthesis involved a combination of artificial gene synthesis and cDNA
cloning to obtain a somatotropin-producing E.coli strain.
 The procedure involves isolating a cloned somatotropin cDNA fragment and cleaving it
with HaeII1, giving DNA encoding 1-24 and 24-191 amino acids.
 The DNA fragments encoding amino acids 24-191 are isolated.
 The human signal sequence is removed because it would not be recognized by the bacterial
secretion machinery.
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 A bacterial signal sequence which specifies secretion of a bacterial protein is ligated in

front of the somatotropin sequence coding for the 24-191 amino acids. This DNA fragment is
then ligated to an expression vector.
 After introduction of the vector into E.coli, somatotropin is produced and the signal
sequence targets the protein for secretion into the space between the inner and outer
bacterial membrane (or the perisplasmic space)
 The protein accumulates in the periplasmic space and is released by hypotonic disruption of
the outer membrane
 Secretion of somatotropin into the perisplasmic space, where there are fewer proteins than
inside the cell make purification simpler.

HOW PLANTS COULD BE PROTECTED FROM TOBACCO MOSAIC VIRUS (TMV)

BY EXPRESSION OF TMV COAT PROTEIN
 The TMV is an RNA virus about 6.5 kb
 A cloned cDNA encoding the coat protein (CP) from TMV is ligated into an integrative T-
DNA vector with a cauliflower mosaic virus (CaMV) promoter
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 The plasmid is transferred into tobacco by Agrobacterium-mediated DNA transfer into leaf
disks
 Regenerated transgenic tobacco plants are obtained that express various levels of TMV coat
protein in their leaves.
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How antisense RNA is used to extinguish gene function.

In antisense technology, the gene to be cloned is ligated into the vector in reverse orientation.
This means that when the cloned gene is transcribed the RNA that is synthesized is the reverse
complement of the mRNA produced from the normal version of the gene. The reverse
complement is referred to as antisense RNA (asRNA). The DNA template strand for a given
mRNA is termed the sense strand. mRNA sequence is complementary to the DNA template
strand from which it is synthesized

Antisense RNA molecules are small (70-110 nucleotides), diffusible transcripts. They pair to
specific complementary target RNAs and control their function and expression by acting as
negative factors or repressors. In most cases, the antisense RNA and the target RNA are
transcribed from opposite strands of the same DNA template by opposing promoters.

Proposed explanations on how antisense RNA functions are:

 Duplex formation with the DNA template blocking transcription
 Blocking intron splicing
 Failure of the antisense:mRNA duplex to be transported to the cytoplasm
 Promoting rapid RNA degradation
 Blocking initiation of translation by preventing attachment of ribosomes to mRNA
 In practice, more than one of these mechanisms may be operating simultaneously.
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Cloning the antisense polygalacturonase gene

During colour and flavour changes associated with the later stages of ripening in tomato, a
number of genes are switched on, including one coding for the enzyme polygalacturonase (see
figure below). This enzyme slowly breaks down the polygalacturonic acid component of the
cell walls in the fruit pericarp, resulting in a gradual softening. The softening makes the tomato
palatable, but if ripening proceeds too much the tomato becomes squashy. Partial inactivation of
the polygalacturonase gene increase the time between flavour development and spoilage of the
fruit. Antisense technology has been used in achieving this as explained below.

A 730-bp restriction fragment was obtained from the 5’ region of the normal polygalacturonase
gene, representing just under half of the coding sequence. A plant polyadenylation signal was
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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. Once inside the plant,
transcription from the cauliflower mosaic virus promoter should result in synthesis of an
antisense RNA complementary to the first half of polygalacturonase mRNA.

Transformation was carried out by introducing the recombinant pBIN19 molecules into
Agrobacterium tumefaciens and then allowing the bacteria to infect tomato stem segments. Small
amounts of callus material collected from the surfaces 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.

The presence of the antisense gene in the DNA of the transformed plants was checked by
Southern hybridization.
Expression of the antisense gene was measured by northern hybridization with a single-
stranded DNA probe that would hybridize only to the antisense RNA.
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The effect of antisense RNA synthesis on the amount of polygalacturonase mRNA in the cells of
ripening fruit was determined by northern hybridization with a second single-stranded probe
specific for the sense mRNA.
The experiments indicated that ripening fruits from transformed plants contained less
polygalacturonase mRNA than the fruits from normal plants.
The amount of polygalacturonase enzyme produced in the repening fruits of transformed plants
were estimated from the intensities of the relevant bands after separation of fruit proteins by
polyacrylamide gel electrophoresis, and directly measuring the enzyme activities in the fruits.

These experiments showed that less enzyme was synthesized in transformed fruits.
The transformed fruits were found to undergo a gradual softening, and could be stored for a
prolonged period before beginning to spoil. This indicated that the antisense RNA had not
completely inactivated the polygalacturonase gene, but had produced a sufficient reduction in
gene expression to delay the ripening process. Therefore the transgenic tomatoes have an
increased shelf-life. They are also resistant to bruising, which simplifies transport of the
tomatoes to the market.

Other applications of antisense RNA

 Artificially constructed antisense RNAs are able to regulate gene expression in prokaryotic
and eukaryotic systems. For example,
 Injection of antisense RNA for the gene encoding actin, a major constituent of the
cytoskeleton, which maintains cell shape, causes the cytoskeleton to disintegrate and the cell
to change their shape.
 Antisense RNA has also been used to suppress two key enzymes in the biosynthesis of the
plant hormone ethylene in order to increase the shelf-life of the tomatoes. Exogenous
ethylene can be used to induce ripening when the need arises.
 Antisense RNA has been used to block the replication of tomato golden mosaic virus in
tobacco plants.
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REPLICATION OF RETROVIRUS AND ITS USE IN GENE THERAPY

Retrovirus replication cycle
 A retrovirus is an RNA virus whose replication depends on formation of a provirus by
reverse transcriptase.

 When the virus enters a cell, its reverse transcriptase makes a double-stranded DNA copy
of the viral RNA
 Reverse transcriptase (or RNA-dependent DNA polymerase is a multifunctional enzyme –
DNA polymerase and ribunuclease actvities

• The enzyme initially binds to a tRNA molecule paired with the end of the viral RNA strand
serving as a primer for the reaction.
• A single strand of complementary DNA (in color) is laid down.

• The enzyme then loops back on itself, and using its ribonuclease activity, degrades the
original viral RNA / while it makes a second DNA strand.
• The final result is a double-strand DNA molecule containing all the viral genetic information.
(see figure below).

• This duplex DNA can then migrate to the nucleus where it is integrated by reverse
transcriptase into host chromosomes to establish a provirus (analagous to a prophage).
• To complete virus replication, host RNA polymerase II makes viral mRNAs, which are then
translated to viral proteins, and full-length RNAs which are packaged into virus particles (see
figure below) that bud out of the infected cell and infects another cell, starting the cycle over
again.

Use of retrovirus in gene therapy

 Gene therapy is a method of curing and inherited disease by providing the patient with a
correct copy of the defective gene.
 There are two basic approaches.
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 (a) In germline therapy, a fertilized egg is provided with a copy of the correct version of
the relevant gene and reimplanted into the mother. If successful, the gene is present and
expressed in all cells of the resulting individual.
 (b) in somatic cell therapy, ordinary cells, usually ones which can be removed from the
organism are transfected and then placed back in the body.
 Gene therapy is most suitable for inherited blood diseases e.g. haemophilia, thalassaemia,
with genes being introduced into stem cells from the bone marrow, which give rise to all
the specialized cell types in the blood.
 A bone extract containing several billion cells, are transfected with retrovirus-based
vector, and then reimplanted into the bone.

 Subsequent replication and differentiation of transfectants leads to the added gene being
present in all the mature blood cells.
 With those genetic diseases where the defect arises because the mutated gene does not
code for a functional protein, all that is necessary is to provide the cell with the correct
version of the gene and the removal of the defective genes is unnecessary.

USING A T-DNA PLASMID TO INTRODUCE A HERBICIDE RESISTANCE GENE

INTO TOBACCO PLANTS
• Herbicide resistance is a useful trait because herbicide resistant plants can survive
treatment while weeds all around it are dying.
• The herbicide glyphosate, active ingredient of weed killer called “Roundup”, kills weeds
by inhibiting an enzyme called EPSP synthase, which is necessary for making the
essential amino acids phenylalanine, tyrosine and tryptophan.
• (EPSP = 5-enol-pyruvylskikimate-3-phosphate).

Procedure for introducing herbicide resistance in tobacco plants

• A mutant EPSP gene is inserted into the T-DNA plasmid under the strong
mannopine synthase promoter.
– The enzyme encoded by the mutant EPSP gene retains its specific activity but
has decreased affinity for the herbicide.
– Hence plants expressing this gene are glyphosate tolerant.
• The recombinant DNA is used to transform Agrobacterium cells.
• The transformed Agrobacterium cells divide repeatedly.
• Disks of 2-mm in diameter are punched out from a tobacco leaf and incubated in
nutrient medium, along with the transformed Agrobacterium cells.
• The cells infect the tabacco tissue and transfer the plasmid bearing the mutant
EPSP gene.
• After the tobacco tissue produces roots around the edge, they are transplanted to
another medium that encourages shoots to form.
• The plants eventually give rise to full-sized tobacco plants.
• The plants are then sprayed with the herbicide to find out whether they are really
resistant.
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THE USE OF VARIABLE NUMBER TANDEM REPEATS (VNTRS) IN DNA

FINGERPRINTING
 Much of the human genome cannot vary greatly between individuals because it has an
essential coding function.
 However, in introns (noncoding regions), changes can be accommodated. One change which
occurs is the tandem repetition of DNA sequences. The tandem repeats are multiple
copies of the same DNA sequence lying in series.
 At each locus, the sequence that is repeated is unique but tends to be GC-rich and 9-40 bp in
length.
 Variation in the number of repeats between unrelated individuals arise from the loss or
gain of the repeat sequence through mutation.
 Tandem repeats which show variable number at different loci and in different
individuals are called VNTRs.
 The VNTR loci in humans are 1-5 kb sequences consisting of variable numbers of a
repeating unit 15 to 100 nucleotides long.
 Because of the variability in the number of tandem repeats from individual to individual, the
set of fragments that shows up on the Southern autoradiogram is highly individualistic.
These patterns are called DNA fingerprints.
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Single locus probes

 The single locus probe is a probe that identifies a single sequence in the genome.
 Diploid organisms therefore usually show two bands in a fingerprint, one allelic variant
from each parent.
 A single locus probe will bind to just one complementary sequence in a haploid genome.
 Single locus probes are fifty times more sensitive than multi-locus probes and can be
used on very small DNA samples.
 They can show a clear profile pattern when as little as 10 -8 g of DNA is present.
 They are also successful in cases where DNA is partially decomposed.
 This is more likely to happen in forensic cases, where warm temperatures and damp
conditions will accelerate the breakdown of DNA.
 Single-locus probes bind to the DNA molecule at one location only and give a pattern of
just two bands per individual.
 There are two bands because an individual has pairs of homologous chromosomes (two
sets of chromosomes, one set from the father and one set from the mother).

Figure…..Genetic fingerprinting of minisatellite DNA sequences

Applications of genetic fingerprinting in forensic science

 Forensic science: procedures related to the scientific tests used to help with police
investigations and legal problems.
 The use of multi-locus DNA profiling in a forensic case is shown in the figure below.
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 In this example, samples of the suspect’s DNA isolated from the victim (V) is the
reference sample.
 Samples from seven suspects were obtained and cut with a restriction enzyme and
separated on an agarose gel.
 The fragments were blotted onto a filter and hybridized with a radioactive probe.
 The probe hybridized to the target sequences, producing a profile pattern when exposed
to X-ray film.
 By matching the band patterns, it is clear that suspect 5 is the guilty person.

Figure……A DNA profile prepared using a multi-locus probe

 The single locus probe will give two bands in the resulting autoradiogram: one from the
paternal chromosome and one from the maternal chromosome.
 In paternity testing (see figure below), samples of DNA from the mother (M), four
children (1-4) and the father (F) were prepared.

 After using a single locus probe, the band patterns showed two maternal bands and two
paternal bands, one band from each homologous chromosome on which the target
sequence is located.
 In the case of child 1, the one band is different from either of the two bands in lane F,
indicating a different father (band labeled DF).
 Child 1 was in fact born to the mother during a previous marriage
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Figure……A DNA profile prepared using a single-locus probe

HEPATITIS B VIRUS VACCINE

Types of Vaccines used prior to recombinant DNA technology
(a) Inactivated vaccines: these are chemically killed derivatives of actual infectious agents.
(b) Attenuated vaccines: these are live viruses or bacteria altered so that they no longer multiply
in the vaccinated person.
 Both types of vaccine work by presenting surface proteins (antigens) to B and T
lymphocytes, destroying the infectious agents before any damage is done.
Problem: these vaccines are potentially dangerous because they can be contaminated with
infectious organisms. For example, there are cases where children have contracted polio fro m
their polio vaccines.
 Recombinant DNA technology has been used for production of subunit vaccines composed
entirely of surface proteins to which the immune system responds. Subunit vaccines are risk-
free.
 The first successful subunit vaccine production was that against hepatitis B virus, which
infects the liver and causes liver damage and sometimes cancer.
 The virus is coated with an antigen HBsAg and infected patients carry large aggregates of
HBsAg in the blood.

Production of recombinant vaccine against HBV in yeast

 Hepatitis B virus (HBV) is encoded by a small 3.2 kb genome that has been cloned and
sequenced.
 The HbsAG gene is ligated to a high-copy yeast expression vector and engineered so that it
would not be secreted.
 The vector is transformed into yeast cells.
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 Transcription occurs from the strong promoter from the gene encoding alcohol
dehydrogenase 1. A transcription terminator is placed downstream.
 The vector contains replication origins and markers for both bacteria and yeast.
 The transformed yeast, Saccharomyces cerevisiae produces large quantities of the viral
protein (about 1-2% of total yeast proteins).
 The transformed yeast can also be grown to high densities (50-100 mg of HbsAg per litre of
culture) in large fermentors.
 The recombinant protein resembles the natural viral protein and forms aggregates with
properties similar to those particles found in HBV-infected patients. Figure…....
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PRODUCTION OF PHARMACEUTICAL PROTEINS IN TRANSGENIC ANIMALS

General information
 Production of therapeutic proteins by mammalian cell culture, where the protein is folded
or processed properly is costly.
 Expression of these proteins in transgenic animals is an alternative method to the costly
technology. For example, secretion of recombinant protein into the milk of transgenic sheep
or cows.
 Milk contains very high levels of proteins such as casein, -lactoglobulin, and whey acidic
protein.
 Production of these proteins is tightly regulated by promoters that limit gene expression only
to cells of the mammary gland.
 The regulatory sequences from the genes of milk-specific proteins have been cloned and
used to control the expression of heterologous genes in transgenic animals.
 The protein is harvested by milking the animals and then standard chromatographic
procedures are used to purify it.
 Examples of pharmacologically active proteins produced by this approach include tissue
plasminogen activator (tPA) (used to dissolve blood clots in humans) and urokinase
normally found in urine (urokinase catalyzes the conversion of plasminogen to plasmin).
Plasmin is a proteolytic enzyme in plasma which can digest many proteins through
hydrolysis.
 Recombinant DNA technology can also be used to produce transgenic cows which produce
milk with an increased concentration of casein, which will in turn give higher yields of
cheese per litre of milk.
 Haemoglobin can also be extracted and purified from red blood cells of transgenic pigs. A
large pig can donate about 20 pints of blood over the course of a year, thereby yielding 500-
1000 grams of purified human haemoglobin.
Procedure for production of pharmaceutically important protein in the milk of transgenic
sheep.
 Your favourite gene (YFG) to be expressed in the transgenic sheep may be the one encodes
the plaminogen activator.
 YFG is placed under the control of the -lactoglobulin promoter, which is only active in
mammalian tissue.
 It is introduced into sheep ova by microinjection of the expression vector into the
pronucleus. A pronucleus is one of the two nuclear bodies of a newly fertilized ovum.
These are male pronucleus and female pronucleus. Their fusion results in the formation of
the zygote.
 The injected ova are implanted into foster mothers and progeny expressing the transgene are
identified by PCR amplification of chromosomal DNA using primers from the sequence of
YFG
 Transgenic sheep express YFG only in mammary tissue and secrete high levels of YFG
protein into the milk, from which it can be purified.
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