GENE EXPRESSION AND REGULATION

Gene expression

Gene expression means how much functional protein is made (if the gene encodes a protein) or
how much of the functional RNA is made (when the gene codes for some functional RNA).

Gene regulation

Gene regulation is all about understanding how cells make decisions about which genes to turn
on, turn off or to tune up or tune down. The knowledge of gene regulation is important for
understanding how cells adjust to changing environments, including how some cells, in
multicellular organisms, decide to become specialized for certain functions (e.g. tissues).

Gene Regulation in microorganisms

The DNA of bacteria and archaea are usually organized into one or more circular chromosomes
in the cytoplasm. The dense aggregate of DNA that can be seen in electron micrographs is called
the nucleoid. In bacteria and archaea, genes, whose expression needs to be tightly coordinated
(e.g. genes encoding proteins that are involved in the same biochemical pathway) are often
grouped closely together in the genome. When the expression of multiple genes is controlled by
the same promoter and a single transcript is produced, these expression units are called operons.
For example, in the bacterium Escherichia coli all of the genes needed to utilize lactose are
encoded next to one another in the genome. This arrangement is called the lactose (or lac)
operon. It is often the case in bacteria and archaea that nearly 50% of all genes are encoded into
operons of two or more genes.

The Role of the Promoter

The first level of control of gene expression is at the promoter itself. Some promoters recruit
RNA polymerase and turn those DNA-protein binding events into transcripts more efficiently
than other promoters. This intrinsic property of a promoter, it's ability to produce transcript at a
particular rate, is referred to as promoter strength. The stronger the promoter, the more RNA is
made in any given time period. Promoter strength can be "tuned" by Nature in very small or very
large steps by changing the nucleotide sequence the promoter (e.g. mutating the promoter). This
results in families of promoters with different strengths that can be used to control the maximum
rate of gene expression for certain genes.

Trp Operon

Logic for regulating tryptophan biosynthesis

E. coli, like all organisms, needs to either synthesize or consume amino acids to survive. The
amino acid tryptophan is one such amino acid. E. coli can either import tryptophan from the
environment (utilizing what it can scavenge from the world around it) or synthesize tryptophan
de novo using enzymes that are encoded by five genes. These five genes are encoded next to

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each other in the E. coli genome into what is called the tryptophan (trp) operon (Figure 1). If
tryptophan is present in the environment, then E. coli does not need to synthesize it and the
switch controlling the activation of the genes in the trp operon is switched off. However, when
environmental tryptophan availability is low, the switch controlling the operon is turned on,
transcription is initiated, the genes are expressed, and tryptophan is synthesized.

Organization of the trp operon

Five genomic regions encoding tryptophan biosynthesis enzymes are arranged sequentially on
the chromosome and are under the control of a single promoter - they are organized into an
operon. Just before the coding region is the transcriptional start site. This is, as the name
implies, the location where the RNA polymerase starts a new transcript. The promoter sequence
is further upstream of the transcriptional start site. A DNA sequence called an "operator" is also
encoded between the promoter and the first trp coding gene. This operator is the DNA sequence
to which the transcription factor protein will bind.

Figure 1: try operon. The five genes that are needed to synthesize tryptophan in E. coli are
located next to each other in the trp operon.

Regulation of the trp operon

When tryptophan is present in the cell: two tryptophan molecules bind to the trp repressor
protein. When tryptophan binds to the transcription factor it causes a conformational change in
the protein which now allows the TF-tryptophan complex to bind to the trp operator sequence.

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Binding of the tryptophan–repressor complex at the operator physically prevents the RNA
polymerase from binding, and transcribing the downstream genes. When tryptophan is not
present in the cell, the transcription factor does not bind to the operator; therefore, the
transcription proceeds, the tryptophan synthesizing genes are transcribed and translated, and
tryptophan is thus synthesized.

Since the transcription factor actively binds to the operator to keep the genes turned off, the trp
operon is said to be "negatively regulated" and the proteins that bind to the operator to silence
trp expression are negative regulators.

The lac operon

Rationale for studying the lac operon

In this example, we examine the regulation of genes encoding proteins whose physiological role
is to import and assimilate the disaccharide lactose, the lac operon. The regulation of the lac
operon is an example of how the coordinated activity of both positive and negative regulators
around the same promoter can be used to integrate multiple different sources of cellular
information to regulate the expression of genes.

The utilization of lactose

Lactose is a disaccharide composed of the hexoses glucose and galactose. It is commonly found
in high abundance in milk and some milk products. As one can imagine, the disaccharide can be
an important food-stuff for microbes that are able to utilize its two hexoses. E. coli is able to use
multiple different sugars as energy and carbon sources, including lactose and the lac operon is a
structure that encodes the genes necessary to acquire and process lactose from the local
environment. Lactose, however, has not been frequently encountered by E. coli during its
evolution and therefore the genes of the lac operon must typically be repressed (i.e. "turned off")
when lactose is absent. Driving transcription of these genes when lactose is absent would waste
precious cellular energy. By contrast, when lactose is present, it would make logical sense for the
genes responsible for the utilization of the sugar to be expressed (i.e. "turned on").

Experiments conducted in the 1950's by Jacob and Monod clearly demonstrated that E. coli
prefers to utilize all the glucose present in the environment before it begins to utilize lactose.
This means that the mechanism used to decide whether or not to express the lactose utilization
genes must be able to integrate two types of information (1) the concentration of glucose and (2)
the concentration of lactose.

The transcriptional regulators of the lac operon

The lac repressor - a direct sensor of lactose

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As noted, the lac operon normally has very low to no transcriptional output in the absence of
lactose. This is due to two factors: (1) the constitutive promoter strength for the operon is
relatively low and (2) the constant presence of the LacI repressor protein negatively influences
transcription. This protein binds to the operator site near the promoter and blocks RNA
polymerase from transcribing the lac operon genes. By contrast, if lactose is present, lactose will
bind to the LacI protein, inducing a conformational change that prevents LacI-lactose complex
from binding to its binding sites. Therefore, when lactose is present the negative regulatory LacI
is not bound to the its binding site and transcription of lactose utilizing genes can proceed.

CAP protein - an indirect sensor of glucose

In E. coli, when glucose levels drop, the small molecule cyclic AMP (cAMP) begins to
accumulate in the cell. cAMP is a common signaling molecule that is involved in glucose and
energy metabolism in many organisms. When glucose levels decline in the cell, the increasing
concentrations of cAMP allow this compound to bind to the positive transcriptional regulator
called catabolite activator protein (CAP). cAMP-CAP complex has many sites located
throughout the E. coli genome and many of these sites are located near the promoters of many
operons that control the processing of various sugars.

In the lac operon, the cAMP-CAP binding site is located upstream of the promoter. Binding of
cAMP-CAP to the DNA helps to recruit and retain RNA polymerase to the promoter. The
increased occupancy of RNA polymerase to its promoter, in turn, results in increased
transcriptional output. In this case the CAP protein is acting as a positive regulator.

Putting it all together: Inducing expression of the lac operon

For the lac operon to be activated, two conditions must be met. First, the level of glucose must
be very low or non-existent. Second, lactose must be present. Only when glucose is absent and
lactose is present will the lac operon be transcribed. When this condition is achieved the LacI-
lactose complex dissociates the negative regulator from near the promoter, freeing the RNA
polymerase to transcribe the operon's genes. Moreover, high cAMP (indirectly indicative of low
glucose) levels trigger the formation of the CAP-cAMP complex. This TF-inducer pair now bind
near the promoter and act to positively recruit the RNA polymerase. This added positive
influence boosts transcriptional output and lactose can be efficiently utilized. The mechanistic
output of other combinations of binary glucose and lactose conditions are descried in the figure 2
that follows.

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Figure 2: lac operon: mechanistic outputs

5
 ISOLATION AND BIOCHEMICAL CHARACTERIZATION OF MUTANTS

The top four techniques used for the isolation of microbial mutants are: Direct Observation,
Enrichment Technique, Replica-Plating Technique and The Ames Test.

1. Direct Observation Technique:

In some cases, a colony growing on an agar plate can easily be seen to be different from the
normal parental type (wild-type). For example, if the parental strain is pigmented, the
observation of non-pigmented colonies may indicate the presence of mutants.

Indicators can also be incorporated into the medium to detect microorganisms with and without
particular metabolic capabilities. For instance, pH indicators can be used into the medium to
detect the production of acidic products. The indication of acid production by one microbial
strain and not by other of the same microorganisms growing under identical conditions would
show the presence of a mutant.

2. Enrichment Technique:

Enrichment technique is employed especially in isolating mutants resistant against phages,

antibiotics, or toxic chemicals. Phage-resistant mutants can be isolated simply by plating the
mutagenized, phenotypically expressed microbial population on plates containing phage
particles. Cells expressing the parental wild-type phenotype are killed; only phage-resistant
mutants develop into colonies. Such colonies are isolated. Similarly, mutants resistant to an
antibiotic or a toxic chemical can be isolated by plating the microbial population with the
antibiotic or the chemical.

3. Replica-Plating Technique:

Replica-plating technique is often used to isolate nutritional mutants (auxotrophs) as well as

various other type of mutants, e.g., antibiotic resistant mutants.

The following steps should be followed when isolating nutritional mutants:

(i) Bacterial cultures are diluted, and the cells are spread on the surface of semisolid nutrient
agar medium in a Petri dish (called “master plate”). The medium in the master plate is a
complete medium i.e., containing all the nutritional components required by the bacterial
population. After a sufficient incubation period, each bacterium produces a visible colony on the
surface of the agar in the master plate.

(ii) A piece of sterile velvet cloth is stretched over a cylindrical block of wood or metal that is
slightly smaller in diameter than the Petri dishes used in the process.

(iii) The master plate is now inverted and gently pressed onto sterile velvet. Since the fibres of
the velvet act as fine inoculating needle, some cells from each colony of the master plate stick to
the velvet.

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(iv) Other Petri dish (called “replica plate”) is taken containing a minimal medium i.e., a medium
deficient with specific nutritional component,

(v) The replica-plate is now inverted and gently pressed onto the velvet thus stamping the
bacterial cells onto the surface of its minimal medium. The replica plate is identically oriented at
the application on the velvet with respect to mark placed on its rim so that the colonies that
appear on the replica plate after incubation occupy positions congruent with those of their
siblings on the master plate.

(vi) After sufficient incubation, it is observed that a colony that develops on the complete
medium of the master plate fails to develop on the minimal medium of the master plate that lacks
a specific nutritional component. Such colony is marked on the master plate and is isolated; it
represents mutant for that specific nutritional component not used in the minimal medium of the
replica plate.

Figure 3: Replica plating technique used for selection of nutritional mutants.

7
The replica-plating technique was developed by Joshua and Esther Lederberg in 1952 in order to
provide direct evidence for the existence of pre-existing mutations originated spontaneously in
microorganisms.

4. The Ames Test:

This test was developed by Ames and coworkers and is based on histidine-requiring (his –)
auxotrophic mutants of Salmonella Typhimurium. Different his– mutants carry different types of
mutations, i.e., transitions, transversions and frame- shifts.

In the Ames test, the frequency of reversion to his+ (prototrophy) is scored in the especially
constructed his– mutants. This is done by placing a known number of mutant cells on medium
lacking histidine and scoring the number of colonies formed. The frequency of cells forming
colonies gives the frequency of reversion. The frequency of spontaneous reversion to his + is quite
rare, i.e., 10-8.

Ames test is routinely used to investigate the mutagenicity of various chemicals. Some of the
chemicals may become mutagenic only when they are acted upon by liver enzymes. For
example, nitrates themselves are neither mutagenic nor carcinogenic. But in eukaryotic cells,
nitrates are converted to introsamines, which are highly mutagenic and carcinogenic. In addition;
some chemicals may be mutagenic only to replicating DNA.

The his– cells are plated onto a medium that contains traces of histidine, which is enough to allow
a few cell divisions, but inadequate for visible colony formation. The test chemical is incubated
with rat liver extract containing the liver enzymes, i.e., the microsomal fraction. This allows
modification of the chemical in the same way as it would be in the liver of animals.

The procedure of Ames test is as follows: The his – bacterial cells are incubated with the liver
extract, and then plated onto a medium containing traces of histidine; this serves as the control
plate. The test plates contain the same medium but the his – cells are not treated with liver extract.
The test chemical is treated with the rat liver extract and a filter paper disc is soaked in this
solution. The filter paper disc is placed onto the medium of test plate.

The chemical present in the filter paper acts on the his – cells growing in the test plate. The
frequency of colonies formed in the control plate and the test plate are compared. An increase in
the frequency in the case of test plate will indicate the test chemical to be mutagenic. In order to
increase the efficiency of the test, the his– strains used in the test are defective in DNA repair,
and have increased permeability to chemicals. It has been observed that more than 90% of the
chemicals that are mutagenic are also carcinogenic.

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Figure 4: The Ames test for mutagenicity. The medium in each petri dish contains a trace of
histidine and a known number of his - cells of a specific Salmonella Typhimurium “tester strain”.
The control plate provides an estimate of the frequency of spontaneous reversion of the tester
strain. The experimental plate shows the frequency of reversion induced by the test chemical.

9
 GENE MAPPING AND GENE MAPS

"Gene mapping" refers to the mapping of genes to specific locations on chromosomes. It is a

critical step in the understanding of genetic diseases. Gene map is a chromosome map that shows
relative locations of genes and other important features. The map is based on the idea of linkage,
which means that the closer two genes are to each other on the chromosome, the greater the
probability that they will be inherited together.

The relative location of two genetic traits can be shown by gene map by using offspring of an
organisms and involves tracking how many times two given genetic traits are inherited together
e.g. hair colour and eye colour. The higher the percentage of descendants that have both traits
together, the closer the genes responsible for the traits will be on the chromosome

There are two types of gene mapping:

Genetic Mapping - using linkage analysis to determine the relative position between two genes
on a chromosome. It requires informative markers (polymorphic) and a population with known
relationships. Genetic mapping uses genetic techniques to indirectly find association between
genes. The unit of distance in genetic maps is centiMorgans, cM (1 cM = 1% chance of
recombination between markers). Genetic mapping offers evidence that a disease transmitted
from parent to child is linked to one or more genes and provides clues about which chromosome
contains the gene and precisely where the gene lies on the chromosome.

Physical Mapping - using all available techniques or information to determine the absolute
position of a gene on a chromosome. Physical mapping relies upon observable experimental
outcomes (hybridization or amplification) and may or may not have a distance measure. Physical
mapping utilizes molecular biology techniques to inspect chromosome directly so that a map
may be constructed with relative gene position.

Physical mapping gives an estimation of the (physical) distance between specific known DNA
sequences on a chromosome. The distance between these known DNA sequences on a
chromosome is expressed as the number of base pairs between them.

There are several techniques used for physical mapping, these include: Restriction mapping
(fingerprint mapping and optical mapping), Fluorescence in situ hybridization (FISH) mapping
and Sequence tagged site (STS) mapping.

The ultimate goal of gene mapping is to clone genes, especially disease genes. Once a gene is
cloned, the DNA sequence can be determine and its protein product studied.

Techniques of gene mapping

1. Gene mapping by in situ hybridization: The method involves hybridizing labeled DNA (or
RNA) probes directly to metaphase chromosomes.

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Figure 5: In situ hybridization using radiolabelled probes

Figure 6: In situ hybridization using non-radiolabelled probes

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 Figure 7: Fluorescence in situ hybridization (FISH)

2. Gene mapping by somatic cell hybridization: The method fuses somatic cells from different
species. It’s an experimentally based segregation technique used to map genes of human
chromosomes. Cells from two different species (e.g. humans and rodents) are artificially
fused together. These culture lines are developed by mixing human and mouse cells in the
presence of the Sendai virus. The virus facilitates the fusing of the two cell types to form a
hybrid cell.
For a reason that is not entirely known, most, but not all, human chromosomes are randomly
lost from the hybrid cell lines. Usually a few human chromosomes are retained. Because the
human and mouse chromosomes can be distinguished by chromosome staining techniques, it
can be determined which human cells are retained with a specific cell line.

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 Exposure
to virus or
to
chemical
agent leads
to cell
fusion

A cell with
two nuclei
forms
(heterokary
on)

The nuclear
membranes
then
fuse

Figure 8: Gene mapping by somatic cell hybridization

3. Gene mapping by gene dosage using patient cells: The method is used to detect dosage
differences in either gene products or gene sequences themselves between patients’ cell lines
containing different numbers of copies of a particular gene. The gene dosage strategy was
originally used to assign genes to chromosome 21 by detecting levels of enzyme activity in
cell lines from patients with Down syndrome that were 1.5-fold higher than levels in cell
lines from chromosomally normal persons. At the DNA level, the dosage approach has been
used increasingly to assign DNA markers to the X chromosome.
4. Gene mapping by chromosomal aberration: Its used to detect directly chromosomal
aberration involving genes which may lead to particular disease.
5. Gene mapping by linkage analysis: Linkage analysis is a method of mapping genes that uses
family studies to determine whether two genes show linkage when passed on from one
generation to the next. Mapping by genetic linkage analysis differs from mapping by physical
methods because physical mapping relies on having a laboratory method to localize a gene
by FISH or by somatic cell hybridization. In contrast, linkage analysis is a tremendously
important and powerful approach in medical genetics because it is the only method that
allows mapping of genes, including disease genes that are detectable only as phenotypic
traits.

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 PRINCIPLE AND APPLICATION OF GENETIC ENGINEERING

Principles of Genetic Engineering

Genetic engineering involves manipulation of the genetic material towards a desired end in a
direct and pre-determined way. This is alternatively called recombinant DNA technology or gene
cloning. It started to develop in the mid-1970s.

Basic steps involved

In short, gene cloning is essentially the insertion of a specific piece of 'desired DNA' into a host
cell in such a way that the inserted DNA is replicated and handed onto daughter cells during cell
division. The main factors involved in gene cloning are the following:

• Isolation of the gene to be cloned.

• Insertion of the gene into another piece of DNA called vector which will allow it to be taken by
bacteria and replicated within them as the cells grow and divide.

• Transfer of the recombinant vector into bacterial cells, either by transformation or by infection
using viruses (transduction).

• Selection of those cells which contain the desired recombinant vectors.

• Growth of the bacteria, that can be continued indefinitely, to give as much cloned DNA as
needed.

• Expression of the gene to obtain the desired product.

1. Isolation of DNA fragments:

The desired DNA fragments can be isolated by means of four mechanisms.

• Restriction endonuclease digestion: It uses the restriction enzymes to cleave the desired
region of the DNA. They are a group of enzymes that recognizes specific nucleotide sequences
in DNA, often 4 or 6 base pairs long, and cut both strands of DNA within the recognition site.
They are site specific.

Two types of cuts are made by these enzymes:

Blunt ends: If it cleaves both DNA strands at precisely opposite points on the two strands, it
leads to blunt end fragments which are difficult to ligate or join to the vector in the next step.

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Cohesive ends: In some cases the two DNA strands are not cut directly at opposite points,
instead, the cuts are staggered forming cohesive ends (sticky ends). Sticky ends are best suited
for cloning purposes as the staggering ends facilitate binding of another piece of DNA.

• Mechanical shearing: It is done by sonication (use of sound waves to shear the DNA) or by
forcing the DNA molecule using a syringe.

• Duplex cDNA synthesis: Sometimes it is possible to synthesize a complimentary DNA

(cDNA) strand to that of the desired DNA. It is done by two methods:

Classical method - here oligonucleotide dT primers, klenow fragment of T4 DNA polymerase

and S1nuclease is used to synthesize cDNA.

New method - here terminal transferase and dCTP primer is used. After removing any
contaminating mRNA by sucrose gradient, oligo dGTP primer is added to synthesize the second
DNA strand.

• Direct chemical synthesis: The desired DNA fragment can be synthesized if the sequence of
the desired DNA is known.

2. Insertion of the desired gene into a vector

Once the desired DNA fragment is obtained it has to be transferred to the host cell. Cloning
vehicles are small plasmids, phage or (animal virus DNA molecules) used to transfer a DNA
fragment into a living cell.

Cloning vehicles are also called vectors. They should have the following properties:

• Origin of replication to enable independent replication.

• Presence of recognition sites for restriction enzymes for insertion of the DNA fragment.

• Must be able to replicate in host cell after transfer.

• Presence of several markers for selection / screening.

The desired gene can be inserted or ligated into the vector by different methods:

Homopolymer tailing: Here same bases are added to the terminal end, for example 8 mol of poly
G tail is added by means of terminal transferase. Thus the complementary strand synthesizes a
poly C tail. So the incoming DNA need not be cut.

Linker molecule: In case of absence of restriction enzymes site, a short sequence which bears a
site for a specific restriction enzyme is introduced and ligated to the DNA by the enzyme DNA
ligase. For non-complementary single strand, linkers are used along with adaptors.

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Blunt end ligation: In case of blunt ends in the DNA and the vector, high concentration of both
plasmid and insert DNA is required and DNA ligase is used to ligate them. Self ligation is found
to occur in low concentration.

Ligation of cohesive terminals: This is more effective and naturally occurring

3. Introduction into the host cell

Once the vector and the desired DNA molecule is ligated, it has to be transferred to a host cell
where it would replicate and produce copies of the desired gene and consequently its products.
This transfer can be achieved by the following methods.

Transfection with recombinant phage DNA: In case the vector being a phage it can infect the
host cell and thus transfer the gene into the host.

Transformation with recombinant plasmid: In case of the vector being a plasmid it can be
transferred to the host by recombination.

4. Selection or screening

After the recombinant DNA is transferred to host via the vector, it is integrated into the host cell
DNA and starts replicating along with the host or is replicated independently along with the
phage within the host. In both the cases the host cells become factories where the desired gene is
replicated and expressed. The step thereafter includes screening of the host cells to check for
successful integration and replication of the desired gene and expression of its products. This is
achieved by the following methods:

Genetic method:

This involves the expression of certain traits. Usually these traits are encoded by the vector or
perhaps by the desired cloned sequence if a direct selection method is available. One of the
simplest methods involves the use of antibiotics to select for the presence of vector molecules.
Eg: pBR322 carries genes Ampr and Tcr which confer resistance to Ampicillin and Tetracycline
respectively.

Screening using nucleic acid hybridization:

It is a very powerful method of screening clone banks, and is one of the key techniques in gene
manipulation. It uses a defined nucleic acid probe which will identify the presence of the desired
gene sequence. The power of nucleic acid hybridization lies in the fact that complementary
sequence will bind to each other with a very high degree of fidelity. Three main types of probes
are used - cDNA , genomic DNA, oligonucleotides.

Immunological screening:

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Here the protein product of a cloned gene is identified by immunological method. Instead of a
nucleic acid probe, a specific antibody is used. Detection may be by radioactive or non-
radioactive method.

Analysis of cloned genes:

This method involves the identification of the protein product by two methods based on
translation of mRNA in vitro. These methods are known as Hybrid release translation (HRT) and
Hybrid arrest translation (HART). HRT is the preferred method.

Blotting techniques:

Here the samples are first made to run in a gel electrophoresis, the separated fragments are then
transferred to a nitrocellulose or nylon membrane by a blotting technique. The original method
uses capillary technique. The filter can then be hybridized with a radioactive probe. After
hybridization the filter is washed and later, exposed to X- ray film and an autoradiogram
prepared, which provides information on the structure of the clone.

*Southern blotting: It is used for running DNA samples. This was first developed by Ed southern
hence the name. Here agarose gel is used.

*Northern blotting: Here RNA samples are been made to run. Here also agarose gel is used.

*Western blotting: It used to find the proteins. Here SDS PAGE method is followed. Membrane
is then probed with an antibody to detect the protein.

Applications of Genetic Engineering

1. Application in Agriculture

An important application of recombinant DNA technology is to alter the genotype of crop plants
to make them more productive, nutritious, rich in proteins, disease resistant, and less fertilizer
consuming. Recombinant DNA technology and tissue culture techniques can produce high
yielding cereals, pulses and vegetable crops.

Some plants have been genetically programmed to yield high protein grains that could show
resistance to heat, moisture and diseases. Some plants may even develop their own fertilizers
some have been genetically transformed to make their own insecticides. Through genetic
engineering some varieties have been produced that could directly fix atmospheric nitrogen and
thus there is no dependence on fertilizers.

Scientists have developed transgenic potato, tobacco, cotton, corn, strawberry, rape seeds that are
resistant to insect pests and certain weedicides. The bacterium, Bacillus thuringiensis produces a
protein which is toxic to insects. Using the techniques of genetic engineering, the gene coding

17
for this toxic protein called Bt gene has been isolated from bacterium and engineered into tomato
and tobacco plants. Such transgenic plants showed resistance to tobacco horn worms and tomato
fruit worms.

There are certain genetically evolved weed killers which are not specific to weeds alone but kill
useful crops also. Glyphosate is a commonly used weed killer which simply inhibits a particular
essential enzyme in weeds and other crop plants. A target gene of glyphosate is present in
bacterium Salmonella Typhimurium. A mutant of Salmonella Typhimurium is resistant to
glyphosate. The mutant gene was then cloned to E. coli and then recloned to Agrobacterium
tumifaciens through its Ti Plasmid. Infection of plants with Ti plasmid containing glyphosate
resistant gene has yielded crops such as cotton, tabacco maize, all of which are resistant to
glyphosate. This makes it possible to spray the crop fields with glyphosate which will kill the
weeds only and the genetically modified crops with resistant genes remain unaffected.

The gene transfer technology can also play significant role in producing new and improved
variety of timber trees. Several species of microorganisms have been produced that can degrade
toxic chemicals and could be used for killing harmful pathogens and insect pests.

Efforts are being made to improve several agricultural crops using various techniques of genetic
engineering which include:

a) Transfer of nitrogen fixing genes (nif genes) from leguminous plants into cereals.
b) Transfer of resistance against pathogens and pests from wild plants to crop plants.
c) Improvement in quality and quantity of seed proteins.
d) Transfer of genes for animal proteins to crop plants.
e) Elimination of unwanted genes for susceptibility to different diseases from cytoplasmic male
sterile lines in crop like maize, where cytoplasmic male sterility and susceptibility are located
in mitochondrial plasmid.
f) Improvement of photosynthetic efficiency by reassembling nuclear and chloroplast genes and
by the possible conversion of C3 plants into C4 plants.
g) Development of cell lines which may produce nutritious food in bioreactors.

2. Application to Medicine

Genetic engineering has been gaining importance over the last few years and it will become
more important in the current century as genetic diseases become more prevalent and agricultural
area is reduced. Genetic engineering plays significant role in the production of medicines.

Microorganisms and plant based substances are now being manipulated to produce large amount
of useful drugs, vaccines, enzymes and hormones at low costs. Genetic engineering is concerned
with the study (inheritance pattern) of diseases in man and collection of human genes that could
provide a complete map for inheritance of healthy individuals.

Gene therapy by which healthy genes can be inserted directly into a person with malfunctioning
genes is perhaps the most revolutionary and most promising aspect of genetic engineering. The

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use of gene therapy has been approved in more than 400 clinical trials for diseases such as cystic
fibres emphysema, muscular dystrophy, adenosine deaminase deficiency.

Gene therapy may someday be exploited to cure hereditary human diseases like haemophilia and
cystic fibrosis which are caused by missing or defective genes. In one type of gene therapy new
functional genes are inserted by genetically engineered viruses into the cells of people who are
unable to produce certain hormones or proteins for normal body functions.

Introduction of new genes into an organism through recombinant DNA technology essentially
alters protein makeup and finally body characteristics.

Vaccines: Recombinant DNA Technology is also used in production of vaccines against

diseases. A vaccine contains a form of an infectious organism that does not cause severe disease
but does cause immune system of body to form protective antibodies against infective organism.
Vaccines are prepared by isolating antigen or protein present on the surface of viral particles.

When a person is vaccinate against viral disease, antigens produce antibodies that acts against
the viral proteins and inactivate them. With recombinant DNA technology, scientists have been
able to transfer the genes for some viral sheath proteins to vaccinia virus which was used against
small pox.

Vaccines produced by gene cloning are contamination free and safe because they contain only
coat proteins against which antibodies are made. A few vaccines are being produced by gene
cloning, e.g., vaccines against viral hepatitis, influenza, herpes simplex virus, virus induced foot
and mouth disease in animals.

Hormones: Until recently the hormone insulin was extracted only in limited quantities from
pancreas of cows and pigs. The process was not only costly but the hormone sometimes caused
allergic reactions in some patients of diabetes. The commercial production of insulin was started
in 1982 through biogenetic or recombinant DNA technology and the medical use of hormone
insulin was approved by food and drug administration (FDA) of USA in 1982. The human
insulin gene has been cloned in large quantities in bacterium E. coli which could be used for
synthesis of insulin. Genetically engineered insulin is commercially available as humilin.

Lymphokines: Lymphokines are proteins which regulate immune system in human body, α -
Interferon is one of the examples. Interferon is used to fight viral diseases such as hepatitis,
herpes, common colds as well as cancer. Such drugs can be manufactured in bacterial cell in
large quantities. Lymphokines can also be helpful for AIDS patients. Genetically engineered
interleukin-II, a substance that stimulates multiplication of lymphocytes is also available and is
being currently tested on AIDS patients.

Somatostatin: A fourteen amino acid polypeptide hormone synthesized by hypothalamus was

obtained only in a small quantity from a human cadavers. Somatostatin used as a drug for certain
growth related abnormalities appears to be species specific and the polypeptide obtained from
other mammals has no effect on human, hence its extraction from hypothalamus of cadavers.

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Genetic engineering technique has helped in chemical synthesis of gene which is joined to the
pBR 322 plasmid DNA and cloned into a bacterium. The transformed bacterium is converted
into somatostatin synthesizing factory. Erythropoetin, a genetically engineered hormone is used
to stimulate the production of red blood cells in people suffering from severe anaemia.

Production of Blood clotting factors: Normally heart attack is caused when coronary arteries
are blocked by cholesterol or blood clot. Plasminogen is a substance found in blood clots.
Genetically engineered tissue plasminogen activator (tPA) enzyme dissolves blood clots in
people who have suffered heart attacks. The plasminogen activator protein is produced by
genetech company which is so potent and specific that it may even arrest a heart attack
underway.

Cancer: Cancer is a dreaded disease. Antibodies cloned from a single source and targeted for a
specific antigen (monoclonal antibodies) have proved very useful in cancer treatment.
Monoclonal antibodies have been target with radioactive elements or cytotoxins like Ricin from
castor seed to make them more deadly. Such antibodies seek cancer cells and specifically kill
them with their radioactivity or toxin.

3. Energy Production

Recombinant DNA technology has tremendous scope in energy production. Through this
technology it is now possible to bioengineer energy crops or biofuels that grow rapidly to yield
huge biomass that used as fuel or can be processed into oils, alcohols, diesel, or other energy
products. The waste from these can be converted into methane. Genetic engineers are trying to
transfer gene for cellulase to proper organisms which can be used to convert wastes like sawdust
and cornstalks first to sugar and then to alcohol.

4. Application to Industries

Genetically designed bacteria are put into use for generating industrial chemicals. A variety of
organic chemicals can be synthesized at large scale with the help of genetically engineered
microorganisms. Glucose can be synthesized from sucrose with the help of enzymes obtained
from genetically modified organisms.

Now-a-days with the help of genetic engineering strains of bacteria and cyanobacteria have been
developed which can synthesize ammonia at large scale that can be used in manufacture of
fertilizers at much cheaper costs. Microbes are being developed which will help in conversion of
Cellulose to sugar and from sugar to ethanol.

Recombinant DNA technology can also be used to monitor the degradation of garbage,
petroleum products, naphthalene and other industrial wastes. For example bacterium
Pseudomonas fluorescens genetically altered by transfer of light producing enzyme called
luciferase found in bacterium Vibrio fischeri, produces light proportionate to the amount of its
breaking down activity of naphthalene which provides way to monitor the efficiency of the
process.

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Maize and soybeans are extensively damaged by black cutworm. Pseudomonas fluorescens is
found in association with maize and soybeans. Bacillus thuringiensis contain a gene pathogenic
to the pest. The pest has, over the years, not only become dangerous to the crops but has
developed resistance to a number of pesticides.

When the gene from B. thuringiensis (Bt) was cloned into Pseudomonas fluorescens and
inoculated into the soil, it was found that genetically engineered Pseudomonas fluorescens could
cause the death of cutworms.

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