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Genetic Recombination

By: Bijaya Kumar Uprety

Contents
Transformation, conjugation, transduction,
protoplast fusion.
Gene cloning and their applications.
Development of hybn'doma for monoclonal
antibodies.
Study of drugs produced by biotechnology
such as Activase, Humulin, Humatrope, and
HB etc.

WORKING GLOSSARY
Auxotroph: A mutated microorganism having nutritional
requirements that differ from those of unmutated microorganisms
from the same strain.
Cloning vector: genetic element into which genes can be
recombined and replicated
Conjugation: transfer of genes from one prokaryotic cell to another
by a mechanism involving cell-to-cell contact and a plasmid
Diploid: a eukaryotic cell or organism containing two sets of
chromosomes
Electroporation: the use of an electric pulse to induce cells to take
up free DNA
Gene disruption: use of genetic techniques to inactivate a gene by
inserting within it a DNA fragment containing an easily selectable
marker. The inserted fragment is called a cassette, and the process
of insertion, cassette mutagenesis

Genetic map: the arrangement of genes on a chromosome


Genome: the total complement of genes of a cell or a virus
Genotype: the precise genetic makeup of an organism
Hybridization: formation of a duplex nucleic acid molecule
with strands derived from different sources by
complementary base pairing
Molecular cloning: isolation and incorporation of a
fragment of DNA into a vector where it can be replicated
Haploid: a cell or organism that has only one set of
chromosomes
Mutagens: agents that cause mutation
Mutant: an organism whose genome carries a mutation
Mutation: an inheritable change in the base sequence of
the genome of an organism

Nucleic acid probe: a strand of nucleic acid that can be


labeled and used to hybridize to a complementary molecule
from mixture of other nucleic acids
Phenotype: the observable characteristics of an organism
Plasmid: an extra chromosomal genetic element that has no
extracellular form
Point mutation: a mutation that involves one or only a very
few base pairs
Polymerase chain reaction (PCR): a method used to amplify
a specific DNA sequence in vitro by repeated cycles of
synthesis using specific primers and DNA polymerase
Recombination: the process by which parts or all of the DNA
molecules from two separate sources are exchanged or
brought together into a single unit.

Restriction enzyme: an enzyme that recognizes and makes


double-stranded breaks at specific DNA sequences
Shotgun cloning: making a gene library by closing random
DNA fragments
Site-directed mutagenesis: a technique whereby a gene
with a specific mutation can be constructed in vitro
Synthetic DNA: a DNA molecule made by a chemical
process in a laboratory
Transduction: transfer of host genes from one cell to
another by a virus
Transformation: transfer of bacterial genes involving free
DNA

INTRODUCTION
Genetic recombination is the process by which genetic elements
from two separate sources are brought together in a single unit.
At molecular level, recombination can be thought of as the
movement of genetic information from one molecule of nucleic
acid to another.
The genetic exchange occurring between homologous DNA
sequences from two different sources is termed as general
recombination. For this to happen, identical sequences on the two
recombining molecules are required.
The process of genetic exchange which occurs in eukaryotes during
sexual reproduction (meiosis) is an example of this type of genetic
recombination.

Figure 1. During meiosis,


homologous
recombination can
produce new
combinations of genes as
shown here
between similar but not
identical copies of human
chromosome 1

Molecular Events in General Recombination


At molecular level, recombination has been studied
only in prokaryotes and viruses.
The process is too complicated to be analysed in the
eukaryotes.
In bacteria, general recombination involves the
participation of a specific protein called the rec A
protein.
The rec A protein is specified by the rec A gene. The rec
A protein is helical in structure and it wraps itself
around the DNA helix, facilitating recombination.
Note: Rec A proteinRec A gene mutation in Rec A
decreased level of genetic recombination.

Bacteria which are mutant in rec A show markedly


decreased levels of general recombination.
An overall molecular mechanism of general
recombination begins with a nick in one of DNA
molecules which leads to the formation of a short
single-stranded segment.
The helix-destabilizing proteins combines at this site
and aids in opening up of the DNA double helix.
The rec A protein binds to the single-stranded fragment
and positions it in such a way that annealing occurs
with a complementary sequence in the adjacent
duplex, simultaneously displacing the resident strand.
Note: nick in one of DNA strand binding of helix
destabilizing proteins rec A proteins binds to single
stranded fragment displacement of resident strand.

Exchange of genetic material occurs between


homologous chromosomes leading to the
formation of recombinant DNA structures.
The means by which DNA fragments are
introduced
into
the
recipient
are:
transformation, transduction and conjugation.

A simplified version of one molecular mechanism of


recombination is shown in figure in the next slide)
Homologous DNA molecules pair and exchange DNA
segments.

The mechanism involves breakage and reunion of


paired segments. Two of the proteins involved, a singlestranded binding (SSB) protein and the RecA protein.
Note that there are two possible outcomes, depending
on which strands are cut during the resolution process.
In one outcome the recombinant molecules have
patches, whereas in the other the two parental
molecules appear to have been cut and then spliced
together.

Detection of Recombination
In order to detect physical exchange of DNA segments, the
cells resulting from recombination must be phenotypically
different from the parents.
Strains that lack some selectable characteristic that the
recombinants will possess. For instance, the recipient may
not be able to grow on a particular medium on which the
genetic recombinants selected can.

Various kinds of selectable and nonselectable markers (such


as drug resistance, nutritional requirements, and so on) may
be used.

Three main processes of genetic recombination in prokaryotes


fragments of homologous DNA from a donor chromosome are
transferred to a recipient cell

(1) Transformation, which involves donor DNA free in


the environment
(2) Transduction, in which the donor DNA transfer is
mediated by a virus
(3) Conjugation, in which the transfer involves cell-tocell contact and a conjugative plasmid in the donor cell

Genetic Transformation
Griffiths Experiment:
Griffith's experiment, conducted in 1928 by Frederick
Griffith, was one of the first experiments suggesting that
bacteria are capable of transferring genetic information
through a process known as transformation.
Griffith used two strains of pneumococcus (Streptococcus
pneumoniae) bacteria which infect mice a type III-S
(smooth) and type II-R (rough) strain. The III-S strain covers
itself with a polysaccharide capsule that protects it from
the host's immune system, resulting in the death of the
host, while the II-R strain doesn't have that protective
capsule and is defeated by the host's immune system. Until
Griffith's experiment, bacteriologists believed that the
types were fixed and unchangeable, from one generation
to another.

In this experiment, bacteria from the III-S strain were killed by heat,
and their remains were added to II-R strain bacteria. While neither
alone harmed the mice, the combination was able to kill its host.
Griffith was also able to isolate both live II-R and live III-S strains of
pneumococcus from the blood of these dead mice. Griffith
concluded that the type II-R had been "transformed" into the lethal
III-S strain by a "transforming principle" that was somehow part of
the dead III-S strain bacteria.
Today, we know that the "transforming principle" Griffith observed
was the DNA of the III-S strain bacteria. While the bacteria had
been killed, the DNA had survived the heating process and was
taken up by the II-R strain bacteria. The III-S strain DNA contains the
genes that form the protective polysaccharide capsule. Equipped
with this gene, the former II-R strain bacteria were now protected
from the host's immune system and could kill the host. The exact
nature of the transforming principle (DNA) was verified in the
experiments done by Avery, McLeod and McCarty and by Hershey
and Chase.

Transformation is a process where certain


competent bacteria are able to take up free DNA
released by other bacteria.
The DNA is taken up only in relatively small amount
and can be acquired only in a single event.
Only certain strains are competent and this ability
seems to be an inherited property of the organism.
Competence in certain bacteria is governed by
certain proteins which include membrane-associated
DNA-binding protein, cell wall autolysin, and various
nucleases.

Increased transformation efficiency in certain species


of bacteria may be due to the deficiency in certain
DNases, which normally destroy incoming DNA.
The nature of the cell surface also plays an important
role in determining whether a cell can take up DNA.
During transformation competent bacteria first bind
DNA reversibly and soon the binding become
irreversible.
Competent cells bind much more DNA than the noncompetent cells (as much as thousand times more).
The transforming DNA is bound at the cell surface by
a DNA- binding protein.

The DNA is either incorporated completely into the cell or is


degraded by the hosts nuclease enzyme, where one strand is
degraded and the other strand is taken up by the host cell.
The incorporated DNA gets associated with a competencespecific protein, which remains attached to the DNA segment
preventing if from the nuclease attack until it reaches the host
chromosome where the rec A protein takes over [rec A is a
product of rec genes (recombination genes).]
The DNA is then integrated into the genome of the recipient
by a recombinational process and rec A proteins are released.

Gene Recombination in Bacteria


Microorganisms carry out several types of gene
recombination, the most common of them being,
general recombination.
This results in a reciprocal exchange of genes
between a pair of homologous chromosomes.
It can occur at any place in the chromosome and
it results from the breakage and reunion of
chromosomes leading to crossing over.
The products of rec genes such as rec-A protein
play an important role in recombination.

In bacterial transformation, a nonreciprocal type of


recombination takes place.
A piece of genetic material is inserted into the
chromosome through the incorporation of a single
strand to form a stretch of heteroduplex DNA.
Another type of recombination, important in the
integration of virus genomes into bacterial
chromosomes, is site-specific recombination.
Transfection is a process where bacteria can be
transformed with DNA extracted from a bacterial
virus rather than from another bacterial cell.

Transfection has become a useful tool for


studying the mechanism of transformation
and recombination because, the small size of
the phage genome allows the isolation of a
nearly homogeneous population of DNA
molecules.

The introduction of DNA into cells


by mixing the DNA and the cell
(a) Binding of free DNA by a membranebound DNA binding protein.

(b) Passage of one of the two strands


into the cell while nuclease activity
degrades the other strand.
(c) The single strand in the cell is bound
by specific proteins, and
recombination with homologous
regions of the bacterial chromosome
mediated by RecA protein occurs.

Transformed cell

The mechanism of bacterial transformation

Agrobacterium tumefaciens Mediated Gene Transfer


What is Agrobacterium tumefaciens?

Bacterial plant pathogen found in the soil that results in tumorous growths and/or roots to
develop in infected plants (Agrobacterium tumefaciens 2001)
This infection is known as Crown Gall Disease (Deacon 2002)

The bacteria transfers a tumor-inducing (Ti) plasmid located in a section of its DNA (known
as T-DNA) into the nucleus of an infected plant cell.

The newly introduced Ti-plasmid is incorporated into the plant genome and is consequently
transcribed (Sforza 2002)

The T-DNA that is integrated into the plant genome contains cancer-causing oncogenic
genes and genes that synthesize opines which are excreted by infected Crown Gall cells and
are a food source for Agrobacterium tumefaciens(Gonzlez-Cabrera 1998)

Transduction
Transduction is a process where DNA is transferred from cell
to cell through the agency of viruses.
Such genetic transfers from the donor to the recipient
through the viruses can occur in two ways, one being
generalized transduction where defective virus particles
randomly incorporate fragments of the host DNA (virtually
any gene of the donor) and transfer it to the recipient cell.
The second is specialized transduction where the DNA of a
temperate virus excises incorrectly and brings adjacent host
genes along with it, and only genes close to the integration
point of the virus are transduced.

The efficiency of generalized transduction is very low


compared to that of a specialized one.
Transduction has been found to occur in a variety of bacteria.
All phages don't participate in transduction and all the
bacteria are not transducible.
But this phenomenon is sufficiently widespread and it plays
an important role in genetic transfer in nature.
Transduction has been found to occur in a variety of
prokaryotes, including certain species of the Bacteria:
Desulfovibrio, Escherichia, Pseudomonas, Rhodococcus,
Rhodobacter, Salmonella, Staphylococcus, and Xanthobacter,
as
well
as
the
archaean
Methanobacterium
thermoautotrophicum.

Generalised transduction
Discovered by Zinder and Lederberg.
This was first extensively studied in the bacterium Salmonella
typhimurium with phage P22
When the population of sensitive bacteria is infected with the
phage, either temperate or virulent, the events of the phage
lytic cycle may be initiated.
In a lytic infection, the host DNA often breaks down into virussized pieces and some of these pieces become incorporated
inside virus particles.
Upon lysis of the cell, these particles are released with the
normal virus particles, so that the lysate contains a mixture of
normal and transducing virus particles.

When this lysate is used to infect a population of


recipient cells, most of the cells become infected
with normal virus particles.
However, a small proportion of the population
receives transducing particles whose DNA can now
undergo genetic recombination with the host DNA.
Since only a small proportion of the particles in the
lysate is of the defective transducing type, the
probability of a defective phage particle transferring
a particular gene is quite low and usually only about
one phage particle in one million transduces a given
marker.

Generalized transduction

In above figure in text box 2 instead of reproduction


its replication.

Generalized transduction: host DNA derived from virtually


any portion of the host genome becomes a part of the
DNA of the mature virus particle in place of the virus
genome.

Specialized transduction: occurs only in some temperate


viruses; DNA from a specific region of the host
chromosome is integrated directly into the virus genome usually replacing some of the virus genes.

Specialized transduction
In contrast to generalized (non-restricted) transduction, which
results in transfer of any gene from donor to recipient
bacterial cell, specialized (restricted) transduction is that
which leads to the transfer of only specific (restricted) genes
from donor to recipient cell.
Specialized transduction is mediated by those temperate
bacteriophages (e.g., lambda (l) phage, mu (m) phage and f80
phage) that usually incorporate (integrate) their DNA into the
bacterial chromosome.
The phage-DNA is called prophage in its integrated state with
the bacterial chromosome; the bacterium having a prophage
is said to be lysogenic, and this phage-host-relationship is
called lysogeny.

Lysogenic temperate phages spontaneously switch over from


lysogenic to lytic state at a low rate (about one in 195 cell divisions)
in nature, or they may be induced to do so by irradiation with
ultraviolet light.
During this transition, the prophage is usually excised precisely from
the specific site of integration in its exactly original form. But
occasionally, it may excise imprecisely so that it takes with it that
specific portion of bacterial chromosome which lies close to the site
of prophage insertion and leaves a portion of its own DNA
remaining integrated within the bacterial chromosome.
Such prophage is called specialized transducing principle and is
packaged into a developing phage particle inside the host bacterial
cell. Phage particle so developed is called specialized transducing
phage and is released after the host bacterial cell undergoes lysis.

Only those specialized transducing phages are viable that


contain an amount of greater than 73% and less than 110% of
the phage-DNA. When a viable specialized-transducing-phage
infects a new bacterial cell, its specialized-transducing
principle that already contains specific portion of bacterial
chromosome inserts into the recipient bacterial chromosome
thus making the latter diploid for that specific bacterial gene
(partial diploid or heterogenote or merogenote). Since the
specialized transducing phage is 'defective' phage as it has lost
some genes during the excision, it functions in recipient
bacterial cell only when the latter is already infected by
another phage (termed as helper phage) that contains the
missing genes.

Conjugation
Bacterial conjugation (mating) is a process of genetic
transfer that involves cell-to-cell contact.
Direct contact between two conjugating bacteria is first made via
a pilus. The cells are then drawn together for the actual transfer
of DNA.

Conjugation involves a donor cell, which contains a


particular type of conjugative plasmid, and a recipient cell,
which does not.
The genes that control conjugation are contained in the tra
region of the plasmid. Many genes in the tra region have to
do with the synthesis of a surface structure, the sex pilus .
Only donor cells have these pili,

The pili make specific contact with a receptor on the


recipient
and then retract, pulling the two cells together.
The contacts between the donor and recipient cells then
become stabilized, probably from fusion of the outer
membranes, and the DNA is then transferred from one cell
to another.

Conjugation involving the transfer of an entire plasmid is the most


common form. The F+ plasmid (F is fertility factor) undergoes rolling circle
replication, meaning that it is replicated as a linear single nucleoside
rather than a complete circular strand of DNA as occurs in replication of
the chromosome. After passing through the pilus into the F- recipient cell,
the complementary nucleoside is synthesized and the plasmid is linked
into circular form by ligase.
Hfr (high frequency recombination) conjugation occurs when a plasmid
from the F+ donor, previously recombined with the chromosome to
produce a new Hfr+ cell, is partially copied along with chromosomal genes
by rolling circle replication and passed to the recipient cell. The DNA
fragment is completed and recombined with the recipient chromosome,
but since all of the plasmid DNA was not transferred, the recipient remains
an F- cell and cannot participate in further conjugation with other cells.

Protoplast fusion

Protoplasts are the cells of which cell walls are removed and cytoplasmic
membrane is the outermost layer in such cells.
Protoplast can be obtained by specific lytic enzymes to remove cell wall.
Protoplast fusion is a physical phenomenon,
During fusion two or more protoplasts come in contact and adhere with
one another either spontaneously or in presence of fusion inducing
agents. By protoplast fusion it is possible to transfer some useful genes
such as disease resistance, nitrogen fixation ,rapid growth rate ,more
product formation rate, protein quality, frost hardiness, drought
resistance, herbicide resistance ,heat and cold resistance from one species
to another.
Protoplast fusion an important tools in strain improvement for bringing
genetic recombinations and developing hybrid strains in filamentous fungi.
Protoplast fusion has been used to combine genes from different
organisms to create strains with desired properties. These are the
powerful techniques for engineering of microbial strains for desirable
industrial properties.

In bacteriology, a protoplast may be defined as the sphere


remaining after Gram-positive bacteria have had their cell
contents lysed ; and the bacterial cell wall constitutes are
absent.
Production of hybrid plants through the fusion of protoplasts
of two different plant species/varieties is called somatic
hybridization and such hybrids are known as somatic hybrids.
The technique of somatic hybridization involves the following
four steps:
1. Isolation of protoplasts
2. Fusion of the protoplasts of desired species/varieties
3. Selection of somatic hybrid cells, and
4. Culture of the hybrid cells and regeneration of hybrid plants
from them.

Significance of Protoplasts Fusion : The various cardinal


significance of protoplast fusion are, namely :
(1) For hybridization between genera or species that are
incapable to cross by the normal and conventional method of
sexual hybridization, and
(2) Significance fully realized in plant kingdom by virtue of the
fact that the hybrid cells are capable of being inducted to
regenerate into whole plants consequently.
Two types of protoplast fusion:
Spontaneous fusion
Induced fusion

Somatic hybridization technique


1. isolation of protoplast

2. Fusion of the protoplasts of desired species/varieties

3. Identification and Selection of somatic hybrid cells

4. Culture of the hybrid cells

5. Regeneration of hybrid plants

Spontaneous fusion
Protoplast during isolation often
fuse spontaneously and this
phenomenon
is
called
spontaneous fusion.
Simply physical contact is
sufficient to bring about the
spontaneous fusion among the
similar parental protoplasts.
During the enzyme treatment for
the isolation of protoplast, it is
found that protoplasts from
adjoining cells fuse through their
plasmodesmata to form a
multinucleate protoplast.

Spontaneous fusion is strictly intraspecific and give


rise to homokaryon.
However, once the protoplasts are freely isolated,
they dont fuse spontaneously with each other.
An exception is the protoplast from microsporocytes
of some plants of lily family where freely isolated
protoplast fuse spontaneously. This type of
spontaneous fusion has been used to produce
intergeneric fusion e.g. the spontaneous fusion of
microsporocyte protoplast of Lolium longiflorum and
Trillium kamtschalicum.

Induced fusion
Fusion of freely isolated protoplasts from different
sources with the help of fusion inducing chemical
agents is known as induced fusion.
Normally, isolated protoplasts dont fuse with each
other as their outer surface carries negative charges
around them and they repel each other.
So this type of fusion needs a fusion inducing agent
or system which actually reduces electronegativity of
the isolated protoplasts and allow them to fuse with
each other.

Various techniques for induced fusion


(a) In animals : Sendai virus (inactivated) is
required to initiate the process of induced
fusion, and
(b) In plants :
PEG treatment ;
NaNO3 treatment;
Calcium ion treatment ;
and electrical impulse are needed to
achieve the phenomenon of induced fusion.

PEG treatment
Polyethylene glycol (PEG) induced protoplast fusion is the
most commonly used as it induces reproducible high
frequency fusion accompanied with low toxicity to most cell
types.
The protoplast mixture is treated with 28-50% PEG for 15-30
min, followed by gradual washing of the protoplasts to
remove PEG; protoplast fusion occurs during the washing.
The washing medium may be alkaline (pH 9-10) and contain a
high Ca2+ ion concentration; this approach is a combination of
PEG and high pH-high Ca2+ treatments, and is usually more
effective than either treatment alone.

PEG is negatively charged and may bind to


cations like Ca2+, which , in turn, may bind to
the negatively charged molecules present in
plasmalemma (plasma membrane).
During the washing process, PEG molecules
may pull out the plasma lemma components
bound to them.
This would disturb plasma lemma organisation
and may lead to the fusion of protoplasts
located close to each other.

Calcium ion treatment


Bhojwani and Razdan* (1983) devised a method involving
centrifugation (spinning) of the protoplasts taken up in a
fusion-inducing solution (0.05 M CaCl2 . 2H2O in 0.4 M
mannitol at pH 10.5) for 30 minutes at 50C,
After which the tubes were incubated at water-bath
maintained at 37C for a duration ranging between 40-50
minutes, which caused fusion of protoplasts to the extent of
20-50%.
However, the method proved to be superior in comparison to
other methods in certain cases, whereas the high pH (10.5)
turned out to be too toxic in other instances.

Electrofusion Technique
It is a more selective and less drastic approach
which utilizes low voltage (65-80 V cm-1)
electric pulses to align the protoplasts in a
single row like a pearl-chain.
The aligned protoplasts can be moved, with a
micromanipulator, and pairs of protoplasts
may
be
isolated
in
individual
microelectrofusion chambers.
The pairs of protoplasts can be fused by a very
brief pulse of high voltage (500-1000 Vcm-1).

Alternatively, the protoplasts may be subjected to


mass electrofusion.
In such case the population of protoplasts is
subjected to high voltage after they are brought
close to each other by the low voltage current.
The high voltage creates transient disturbances in
the organisation of plasma lemma, which leads to
the fusion of neighbouring protoplasts.
The entire operation is carried out manually in
specially designed equipment, called electroporator.

Fusion induced by Sodium or Potassium Nitrate


In this method, equal densities of protoplast from two
different sources are mixed and then centrifuged at 100 g
for 5 mins to get a dense pellet.
This is followed by addition of 4 ml of 5.5% sodium
nitrate in 10.2% sucrose solution to resuspend the
protoplast pellet.
The suspended protoplasts are kept in waterbath at 350C
for 5 mins and again centrifuged at 200g for 5 mins.
[g = (1.118 10-5) R S2]; where g is relative centrifugal
force, R is radius of rotor and S is speed of centrifuge in
revolution per minute (RPM).

The pellet is once again kept in waterbath at 30 0C for 30


mins.
Fusion of protoplast takes place at the time of incubation.
The pellet is again suspended by 0.1% sodium nitrate for 5-10
mins.
The protoplasts are washed twice with liquid culture medium
by repeated centrifugation.
Finally, the protoplasts are plated in semisolid culture
medium.
The frequency of fusion is not high in this method and sodium
nitrate is toxic to cell at high concentration yet this is one of
the useful technique for protoplasts derived from
meristematic cells.

What is cloning????
Cloning in biology is the process of producing
similar populations of genetically identical
individuals that occurs in nature.
Cloning in biotechnology refers to processes
used to create copies of DNA fragments
(molecular cloning), cells (cell cloning),
or organisms.

What is DNA cloning?


When DNA is
extracted from an
organism, all its
genes are obtained
In gene (DNA)
cloning a particular
gene is copied
(cloned)

What is Gene cloning?


To "clone a gene" is to make many copies of it.
Act of making many identical copies of gene.

Gene can be an exact copy of a natural gene.


Gene can be an altered version of a natural
gene.
The term gene cloning covers a wide range of
techniques that make it possible to manipulate
DNA in a test tube and also to return it to living
organisms where it functions normally.

Restriction Enzymes
Bacteria have learned to "restrict" the possibility of attack
from foreign DNA by means of "restriction enzymes.
Cut up foreign DNA that invades the cell.
Type II and III restriction enzymes cleave DNA chains at
selected sites.
Enzymes may recognize 4, 6 or more bases in selecting
sites for cleavage.
An enzyme that recognizes a 6-base sequence is called a
"six-base cutter.

Type II restriction enzyme nomenclature


Why the funny names?

EcoRI
BamHI
DpnI
HindIII
BglII
PstI
Sau3AI
KpnI

Escherichia coli strain R, 1st enzyme


Bacillus amyloliquefaciens strain H, 1st enzyme
Diplococcus pneumoniae, 1st enzyme
Haemophilus influenzae, strain D, 3rd enzyme
Bacillus globigii, 2nd enzyme
Providencia stuartii 164, 1st enzyme
Staphylococcus aureus strain 3A, 1st enzyme
Klebsiella pneumoniae, 1st enzyme

Figure 1.1 The basic steps in gene cloning.

Ever since the British scientists, in 1997, carried out the


successful cloning of sheep (named DOLLY) by meticulously
transferring the nucleus from an udder-cell of an adult sheep
right into the cytoplasm of an enucleated fertilized egg.
Subsequently, the resulting egg was neatly transplanted into
the uterus of a surrogate mother wherein it eventually
developed just like a normal zygote
Ultimately into a normal lamb that has now grown into a
normal adult sheep.
Therefore, one may rightly conclude and infer, based on the
above actual realistic experimental evidences, that
when complete animals are duly accomplished from the
somatic cells of an animal it is usually termed as animal
cloning.

Cloning of Dolly (eg of animal cloning)

Cloning of dolly

Cloning process
(i) DNAcloning,
(ii) Cloning larger DNA fragments in specified cloning vectors,
(iii) Cloning Eukaryotic DNAs in bacterial plasmids,
(iv) Cloning Eukaryotic DNAs in phage genomes,
(v) Cloning cDNAs
(vi) Expression cloning.
(vii) Amplifying DNA : The Polymerase Chain Reaction (PCR)

DNA-cloning
The DNA cloning is nothing but a broad based technique whereby large
amount of a particularly DNA-segment are produced.
Usually, the DNA segment which is to be cloned is first linked to a vector
DNA, that serves as a vehicle for carrying foreign DNA into a suitable host
cell, such as the bacterium Escherichia coli.
The vector (i.e., E. coli) essentially contains sequences which in turn
permits to be replicated within the host cell. In order to clone DNAs within
bacterial hosts two types of vectors are commonly employed, namely :
(a) The DNA segment to be cloned in introduced into the bacterial cell by
first joining it to a plasmid and secondly, causing the bacterial cells to take
up the plasmid from the medium, and
(b) The DNA segment is joined to a portion of the genome of the bacterial
virus lambda () which is subsequently allowed to infect a culture of
bacterial cells. Thus, a huge quantum of viral progeny are produced, each
of which contains the foreign DNA segment.

By following either of the two methods stated above


techniques the DNA segment once gets inside a bacterium, it
will undergo the replication process with the bacterial (or
viral) DNA and partitioned to the daughter cells (or progeny
viral particles).
In this manner, the actual number of bacterial cells which are
actually formed.
Besides, cloning may also be employed as a versatile method
to isolate in a pure form any specific DNA fragment amongst a
relatively large heterogeneous population of DNA molecules.

Cloning larger DNA fragments in specified cloning vectors


It has been observed that neither plasmid or lambda phage () vectors are
adequately suitable for cloning DNAs whose length is more than 20-25 kb.
This has revitalized the interest of researchers to look into the
development of several other vectors which might facilitate to clone much
larger segments of DNA. However, the most important to these vectors
are termed as yeast artificial chromosomes (YACs).
YACs are nothing but artificial versions of a normal yeast chromosome.
They normally comprise of all the elements of a yeast chromosome which
are absolutely necessary for the specific structure to be replicated during
S-phase and subsequently segregated to daughter cells during mitosis,
including :
One of more origins of replication,
Having telomers at the ends of the chromosomes, and
A centromere to which the spindle fibers may get attached during
chromosome separation.

Invariably, the YACs are designed in such a fashion so as to provide


essentially :
(a) A gene whose encoded product permits those particular cells having the
YAC to be selected from those that lack the element, and
(b) The DNA fragment to be cloned like other cells, subsequently the yeast cells
shall pick up DNA from their respective medium that caters for the path
whereby YACs are introduced directly into the cells.
It has been observed that DNA fragments cloned in YACs range typically
from 100kb to 1,000 kb in length. Example : The restriction enzyme
usually recognizes the eight-nucleotide sequence GCGGCCGC, which in
turn specifically cleaves mammalian DNA into fragments approximately
one million base pairs long. Fragments of this length can now be
introduced conveniently into YACs and subsequently cloned within host
yeast cells.

Fig. Strategy for using YAC

Cloning eukaryotic DNA in bacterial plasmid


A foreign DNA intended to be cloned is strategically inserted
into the plasmid to give birth to a recombinant DNA molecule.
However, the plasmid used for DNA cloning are exclusively the
modified versions of those occurring in the bacterial cells.
Consequently, the bacterial cells are able to take up DNA
from their medium. This particular phenomenon is termed as
transformation and forms the basis for cloning plasmid in
bacterial cells.
Fig. 2.8. represents the DNA cloning using bacterial plasmid.
First of all the recombinant plasmids each containing a
different foreign DNA insert are added to a bacterial culture
(E. coli) which has been previously treated with Ca2+ ions.

These bacteria are gainfully stimulated to take up DNA from


their respective surrounding medium upon exposure to a brief
thermal-shock treatment yielding plasmid DNA (purified).
Secondly, human DNA are also obtained in the purified form.
Subsequent treatment of human DNA and plasmid DNA with
EcoR1 result into the cleavage of human and bacterial DNA
into various sized fragments.
Now, these small fragments join together to yield
recombinant DNAs with DNA ligase and thus give rise to the
plasmids. These population of plasmids invariably contain
various segments of human DNA.
Incubation of these plasmids with E. coli cells under controlled
experimental parameters ultimately yields plasmid that are
free from E coli.

It has been observed that only a very small


percentage of the cells are competent to pick up and
retain one of the combinant replicate molecules.
Once it is taken up the plasmid undergoes replication
autonomously within the recipient and is
subsequently passed on to its progeny during cell
division.
The isolated recombinant plasmids can then be
treated with the same restriction enzymes used in
their formation, that releases the cloned DNA
segments from the remainder of the DNA which
served as the vector. Thus, the cloned DNA can be
separated from the plasmid.

Cloning eukaryotic DNAs Phage genome

A bacteriophase, or more commonly a phage is a virus particle which infects a


bacterial cell.
In fact, a phage particle normally comprises of two essential components ; first, a
phage head that contains the genetic material and secondly, a tail through which
the genetic material is injected into the host cell.
Interestingly, one of the most broadly explored of the these phage particles,
termed Bacteriophage Lambda [or bacteriophage ()], has more or less turned out
to be a commonly employed cloning vector.
The genome of lambda is a linear and double-stranded DNA molecule having 50kb
length.
In usual practice, the modified strain (mutant) employed in most cloning
experiments contains two cleavage sites for the enzymes EcoR1 that ultimately
fragments the genome into three large segments.
However, the two outer segments essentially contain all informations required for
the infectious growth, whereas the middle fragment could be rejected
conveniently and replaced suitably by a piece of DNA upto 25kb in length.

The two outer segments of the bacteriophage undergo splicing with


eukaryotic fragment to result into the formation of recombinant DNA.
Consequently, the recombinant DNA molecules can be packaged into
phage heads in vitro and in turn these genetically engineered phage
particle may be employed to infect host bacteria.
Once gaining entry into the bacteria, the eukaryotic DNA segment is
adequately amplified along with the viral DNA and subsequently packaged
into an altogether new generation of virus particle that are released when
the cell undergoes lysis.
The released particle thus obtained infect new cells, and without any loss
of time either a plaque (A zone of lysis or cell inhibition caused by virus
infection on a lawn of cells) or a clear spot in the bacterial lawn is visible
distinctly at the site of infection. Each plaque, which is nothing but a zone
of lysis, possesses millions of phage particle, each carrying a single copy of
the same eukaryotic DNA segment.)

Fig. Sequence for cloning DNA fragments in Lambda (I)


phage.

Cloning cDNA

The central dogma of molecular biology outlines that in synthesizing


proteins, DNA is transcribed into mRNA, which is translated into protein. One
difference between eukaryotic and prokaryotic genes is that eukaryotic genes
can contain introns (intervening sequences) which are not coding sequences (in
contrast with exons which are coding sequences), and must be removed from the
RNA primary transcript before it becomes mRNA and can be translated into
protein. Prokaryotic genes have no introns, so their RNA is not subject to splicing.

Often it is desirable to express eukaryotic genes in prokaryotic cells. A simplified


method of doing so would include the addition of eukaryotic DNA to a vector,
sometimes a prokaryotic host, which would then transcribe the DNA to mRNA
and then translate it to protein. However, as eukaryotic DNA has introns, and
since prokaryotes lack the machinery to splice them, the splicing of eukaryotic
DNA must be done prior to adding the eukaryotic DNA into the host. This DNA,
which was made as a complementary copy of the RNA and has no introns, is
called complementary DNA (cDNA). To obtain expression of the protein encoded
by the eukaryotic cDNA, prokaryotic regulatory sequences would also be
required (e.g. a promoter).

The cloning that has been described here will work for any random piece of DNA.
But since the goal of many cloning experiments is to obtain a sequence of DNA
that directs the production of a specific protein, any procedure that optimizes
cloning will be beneficial. One such technique is cDNA cloning.

The principle behind this technique is that an mRNA population isolated from a
specific developmental stage should contain mRNAs specific for any protein
expressed during that stage. Thus, if the mRNA can be isolated, the gene can be
studied.

mRNA cannot be cloned directly, but a DNA a copy of the mRNA can be cloned. (In
this regard, the term cDNA is short for "copy DNA".) This conversion is
accomplished by the action of reverse transcriptase and DNA polymerase. The
reverse transcriptase makes a single-stranded DNA copy of the mRNA.

The second DNA strand is generated by DNA polymerase and the double- stranded
product is introduced into an appropriate plasmid or lambda vector.

In order to clone cDNAs, first of all a sizable population of


mRNA is isolated ;
secondly, it is employed as a template to provide a singlestranded DNA complement ;
thirdly, the resulting product (single stranded) is duly
converted to a double stranded population with the help of a
DNA polymerase ;
and fourthly, they are finally combined with the desired vector.
It is quite evident that essentially mRNA populations typically
consists thousands of altogether different species, and as with
experiments employing genomic DNA fragments, the clones
should be invariably screened to isolate only one particular
sequence from a heterogeneous population of recombinant
molecule.

it may be observed that when


polypeptide (A) and mRNA are
annealed, it provides a small
segment of primer attached to
poly (A) to the tail of mRNA. Now,
with the help of reserve
transcriptase the primer to poly
(A) gets fully developed. Alkali
helps in the separation of DNA
and RNA strands to give rise to
fully developed primer alone,
which on treatment with RNA
polymerase 1 yields the combined
product. The resulting product
when digested with S1 nuclease
two separate strands of the
primer and poly (A) are obtained.
Lastly, integrate cDNA into the
plasmid vector that will produce
a bacterium wherein DNA can be
cloned.

Fig: Sythesis whereby cDNA


get cloned in plasmid.

Expression cloning
Expression cloning is a technique in DNA cloning that uses expression
vectors to generate a library of clones, with each clone expressing one
protein.
This expression library is then screened for the property of interest and
clones of interest recovered for further analysis.
An expression vector is a relatively small DNA molecule that is used to
introduce and express a specific gene into a target cell. Once the
expression vector is inside the cell, the protein that is encoded by the
gene is produced by the cellular transcription and translation machinery.
Usually the ultimate aim of expression cloning is to produce large
quantities of specific proteins.
Expression vectors are used for molecular biology techniques such as sitedirected mutagenesis. In general, DNA vectors that are used in many
molecular biology gene cloning experiments need not result in the
expression of a protein.

Expression vectors are often specifically designed to contain regulatory


sequences that act as enhancer and promoter regions, and lead to
efficient transcription of the gene that is carried on the expression vector.
Expression vectors are basic tools for biotechnology and the production of
proteins such as insulin that are important for medical treatments of
specific diseases like diabetes.
Expression vectors are used for molecular biology techniques such as sitedirected mutagenesis. In general, DNA vectors that are used in many
molecular biology gene cloning experiments need not result in the
expression of a protein.
Expression vectors are often specifically designed to contain regulatory
sequences that act as enhancer and promoter regions, and lead to
efficient transcription of the gene that is carried on the expression vector.
Expression vectors are basic tools for biotechnology and the production of
proteins such as insulin that are important for medical treatments of
specific diseases like diabetes.

:::::Note:::::

All vectors used for propagation of DNA inserts in a suitable host are called
cloning vectors.
But when a vector is designed for the expression of i.e. production of the protein
specified by, the DNA insert, it is termed as expression vector.
As a rule, such vectors contain at least the regulatory sequences, i.e. promoters,
operators, ribosomal binding sites, etc, having optimum function in the chosen
host.
There
are
many
types
of
cloning
vectors.
Genetically
engineered plasmids and bacteriophages (such as phage ) are perhaps most
commonly used for this purpose. Other types of cloning vectors include bacterial
artificial chromosomes (BACs) and yeast artificial chromosomes(YACs).
In the case of expression vectors, the main purpose of these vehicles is the
controlled expression of a particular gene inside a convenient host organism
(e.g.E. coli). Control of expression can be very important; it is usually desirable to
insert the target DNA into a site that is under the control of a
particular promoter. Some commonly used promoters are T7
promoters, lac promoters (bla promoter) and cauliflower mosaic virus's 35s
promoter (for plant vectors).

Applications
Protein Production- An expression vector with
protein producing DNA is imported into a cell
through the use of plasmids into a bacterial
cell. This causes the bacterial cell to then
produce said protein. This method can be
used in medicine to farm proteins on a widescale such as Insulin, Erythyroprotein, or
Tissue Plasminogen Activator.

In genetics, a promoter is a
region of DNA that facilitates
the
transcription
of
a
particular gene. Promoters are
located near the genes they
regulate, on the same strand and
typically upstream (towards
the 5' region of the sense
strand).

An enhancer is a short region


of DNA that can be bound
with proteins (namely, the transacting factors, much like a set
of transcription factors) to
enhance transcription levels
of genes (hence the name) in
a gene cluster.
Fig. The creation of an expression vector with which proteins
can be grown.

Amplifying DNA: The polymerase chain reaction (PCR)


Gene Amplification through Polymerase chain reaction
The polymerase chain reaction is one of the powerful gene
amplification technique developed by Kary Mullis in 1985.
It generates microgram quantities (upto billion copies) of DNA
copies of the desired DNA (or RNA) segment, present even as
a single copy in the initial preparation, in a matter of few
hours.
The PCR utilizes the following:
1. DNA preparation containing the desired segment to be
amplified
2. Two nucleotide primers (about 20 bases long) specific, i.e.
complementary, to 3- borders (the sequences present at the
3- ends of the two strands) of the desired segment,

The four deoxynucleoside triphosphates


[ dTTP (deoxythymidine
triphosphate), dCTP (deoxycyctidine triphosphate), dATP (deoxyadenosine
triphosphate) and dGTP (deoxyguanosine triphosphate), and a heat stable
DNA polymerase, e.g. Taq (isolated from the bacterium Thermus
acquaticus), pfu (from Pyrococcus furiousus) and Vent (from Thermococcus
litoralis) polymerases. Pfu and Vent polymerases are more efficient than
the Taq polymerase.
Procedure of PCR:
At the start of PCR, the DNA from which a segment is to be amplified, an
excess of the two primer molecules, the four deoxynucleoside
triphosphates and the DNA polymerase are mixed together in the reaction
mixture that has appropriate quantities of Mg2+
The following operations are now performed sequentially:
Denaturation:
The reaction mixture is first heated to a temperature between 90-98 0C
(commonly at 94 0C) that ensures DNA denaturation. This is the
denaturation step. The duration of this step in the first cycle of PCR is
usually 2 min at 94 0C.

Annealing:
The mixture is now cooled to a temperature (generally, between 40-60 0C)
that permits annealing of the primer to the complementary sequences in
the DNA. As a rule, these sequences are located at the 3 ends of the two
strands of the segment to be amplified. This step is called annealing.
The duration of annealing step is usually 1 min during the first as well as
the subsequent cycles of PCR. Since the primer concentration is kept very
high relative to that of the template DNA, primer-template hybrid
formation is greatly favoured over reannealing of the template strands.
Primer Extension:
The temperature is now so adjusted that the DNA polymerase synthesizes
the complementary strands by utilizing the 3-OH of the primers, this
reaction is the same as that occurs in vivo during replication of the leading
strand of a DNA duplex.
The primers are extended towards each other so that the DNA segment
lying between the two primers is copied.
The duration of primer extension is usually 2 mins at 72 0C.

The completion of the extension step completes the


first cycle of amplification; each cycle may take few
minutes.
It should be noted that the extension of primer
continues till the strands are separated during the
denaturation step of the next PCR cycle.
The product of first cycle is the long product.
Numerous subsequent cycle takes place similar to
first cycle thus amplifying the the gene exponentially.

Development of hybridoma for monoclonal antibody


(Mas)
A hybridoma is a hybrid cell obtained by fusing
antibody producing cell and a multiple myeloma
cell. (Multiple myeloma is a cancer of plasma cell.)
OR It may be defined as a hybrid cell obtained by
fusing B-lymphocyte with usually a tumor cell of the
antibody forming system or of B-lymphocytes (these
are called myelomas).
The hybrid cells thus produced possess the ability to
produce
antibodies due to the B-lymphocyte
genome and the capacity for indefinite growth in
vitro due to the tumor (myeloma) cell involved in the
fusion.

Therefore, hybridoma cells are either cultured in


vitro or passaged through mouse peritoneal cavity to
obtain monoclonal antibodies. This is called
hybridoma technology.
B-lymphocytes are isolated from the spleen of an
animal, e.g, mouse, which had been immunized with
the antigen against which monoclonal antibodies are
to be raised (produced).
Myeloma cells are selected for mainly two features:
(1) these cells must not produce antibodies
themselves, and

(2) they must contain a genetic marker e.g.


HGPRT- trait (hypoxanthine guanine
phospho-ribosyltransferase), which permits an
easy selection of the resulting hybrid cells.
When HGPRT- cells are fused with Blymphocytes, the resulting cell population will
consist of (1) hybrid cells (hybridomes), (2)
myeloma cells (3) B-lymphocytes.
This cell population is now cultured in HAT
medium containing the drug aminopterin.

Similarly, the B-lymphocytes do not grow for long


periods of time in tissue culture and eventually die.
In contrast, only the hybridoma cells proliferate on
the HAT medium since the B-lymphocyte genome
makes them HGPRT+ and they have the capability for
indefinite growth from the myeloma cell.
Thus hybridomas (myeloma +B-lymphocyte hybrid
cells) are selected by using a suitable selective
medium like HAT, which allows only the hybridomas
to proliferate.

The next step consists of identification and isolation of the


hybridoma cells producing antibodies specific to the antigen
used for immunization of the animals.
[The hybridoma cells are suspended, suitably diluted and
distributed into micro-wells, one cell in each micro-well, and
allowed to grow. The hybridoma cells grow and secrete
antibodies into the medium. The supernatant from each
micro-well is sampled and assayed for the presence of
antibodies specific to the antigen in question using one of the
assay methods e.g. ELISA. Wells containing the antibodies
specific to the antigen are identified and the hybridoma cells
from them isolated and cloned to ensure that a hybridoma
clone produces antibodies of a single specificity.]

Once the desired hybridoma clone has been


obtained, it is multiplied either in vitro or in
vivo to obtain monoclonal antibodies.
In vivo production system involves injection of
hybridoma cells into the peritoneal cavity of
isogenic animals, collection of the ascitic fluid
and separation of the antibodies from it.
In vitro production involves growing
hybridoma cells are grown in vitro in a suitable
large scale culture system and the monoclonal
antibodies are purified from these cultures.

HAT medium

HAT medium is one of the several selective media used for the
selection of hybrid cells.
This medium is supplemented with hypoxanthine,
aminopterin and thymidine, hence the name HAT medium.
Antimetabolite aminopterin blocks the cellular biosynthesis of
purines and pyrimidines from simple sugars and amino acids.
However, normal human and mouse cells can still multiply as
they can utilize hypoxanthine and thymidine present in the
medium through a salvage pathway, which ordinarily recycles
the purines and Pyrimides produced from degradation of
nucleic acids.

Hypoxanthine is converted into guanine by the enzyme


HGPRT, while thymidine is phosphorylated by thymidine
kinase (TK); both HGPRT and TK are enzymes of the salvage
pathway.

On a HAT medium, only those cells that have active HGPRT


(HGPRT+) and TK (TK+) enzymes can proliferate, while those
deficient in these enzymes (HGPRT- and/or TK-) cannot divide.
Thus , one may fuse HGPRT deficient human cells (designated
as TK+ HGPRT-) with TK deficient mouse cells (denoted as TKHGPRT+). Their fusion products (hybrid cells) will be TK+ (due
to human gene) and HGPRT+ (due to mouse gene) and will
multiply on the HAT medium , while the man and mouse cells
will fail to do so.

Application of monoclonal antibodies (Mabs)


Diagnostic application:
When Mabs are used to detect the presence of
a specific antigen or of antibodies specific to
an antigen in a sample or samples, this
constitutes a diagnostic application. Some
examples of diagnostic applications are as
follows:
1. Mabs are available for the unequivocal
classification of blood groups e.g. ABO, Rh,
etc.

2. Mabs are applied for a clear and decisive detection


of pathogens involved in disease (disease diagnosis).
3. Mabs can be used for the accurate detection of
specific chromosomes of a given species.
Therapeutic application:
1. Antibodies specific to a cell type, say, tumor cells,
can be linked with a toxin polypeptide to yield a
conjugate molecule called immunotoxin. The
antibody component of immunotoxin will ensure its
binding specifically and only to the target cells and
the attached toxin will kill such cells.

2. Mabs can be administered to provide passive


immunity against diseases.
3. Mabs are very useful in the purification of
antigens specific to pathogens; these purified
antigens are used as vaccines.
Immunopurification:
The highly specific interaction of an antibody
to the antigen is used to purify antigens
present in small quantities as a mixture with
several types of molecules; this is known as
immunopurification.

Drugs produced by biotechnology

The European Federation of Biotechnology (FEB) considers


biotechnology as the integration of natural sciences and
organisms, cells, parts thereof, and molecular analogues for
products and services.

New Biotechnological processes essentially embrace almost


all methods of genetic modification by recombinant DNA and
cell fusion techniques, together with the magic touch of the
modern developments of the so-called traditionalbiotechnological processes.
Interestingly, these processes will, in many instances, function
at relatively low temperature, will consume little energy, and
will rely mainly on inexpensive substrates for biosynthesis.

Although there are a large number of drugs that


have been evolved via the biotechnological
processes. Few of which are listed below:
(i) Altepase [Activase],
(ii) Human Insulin [Humulin(R)],
(iii) Humatrope : Growth Hormone, and

(iv) Hepatitis B [Recombinant HB (Merck) a Hepatitis


B vaccine]

Alteplase [ Activase]
Activase (Alteplase) is FDA-approved for treatment of myocardial
infarction (heart attack), acute ischemic stroke (blood clot in the
brain) & acute massive pulmonary embolism (blood clots in the
lungs).
Activase is one of the recombinant tissue plasminogen activator, or
r-tPA and is produced by recombinant DNA technology.
For certain patients, Activase may improve the chances of recovery
from stroke with little or no disability. Patients can receive Activase
only if they begin treatment within 3 hours after their stroke
symptoms start and only after they have had a scan to rule out
bleeding in the brain.
Tissue plasminogen activator (abbreviated tPA or PLAT) is
a protein involved in the breakdown of blood clots. It is a serine
protease found on endothelial cells, the cells that line the blood
vessels.

As an enzyme, it catalyzes the conversion of plasminogen to plasmin, the


major enzyme responsible for clot breakdown. Because it works on
the clotting system, tPA is used in clinical medicine to treat only embolic or
thrombotic stroke. Use is contraindicated (not advisable) in hemorrhagic
stroke and head trauma.

tPA may be manufactured using recombinant biotechnology techniques.


tPA created this way may be referred to as recombinant tissue
plasminogen activator (rtPA). Recombinant tissue plasminogen activators
(r-tPAs) include alteplase, reteplase, and tenecteplase (TNKase).
Storage: Alteplase need to be stored preferably at 20C or even below in
perfectly sealed containers.

Units : The activity of alteplase may be measured in terms of


International Units (IU) by employing the 2nd International
Standard for the Tissue Plasminogen Activator established in
1987, although it is an usual practice to express the doses by
weight. The Specific Activity of alteplase is 580 000 IUs. mg 1.
Pharmacokinetics : It has been duly observed that alteplase
gets cleared from the plasma, chiefly via metabolism in the
liver.
Note: IU is the amount of an enzyme that catalyses the transformation of 1 micromole of substrate per
minute (under defined conditions of pH, concentration, and temperature)

Uses and Mechanism of Action :


The various applications and possible mechanism of action of
alteplase are as follows :
(1) It is a thrombolytic agent, which is a predominant representative
of a single-chain form of the endogenous enzyme tissue
plasminogen activator meticulously produced by the recombinant
DNA technology.
Very much similar to the endogenous tissue plasminogen activator,
it converts fibrin-bound plasminogen to the corresponding active
form of plasmin, thereby causing in marked and pronounced
fibrinolysis and dissolution of clots.
(2) Alteplase is employed very much akin (similar in quality and
character)
to steptokinase both in the management and
treatment of thrombo-embolic disorders, specifically the
myocardial infarction and venous thrombo-embolism.

(2) Alteplase has almost negligible effect upon the circulating,


unbound plasminogen ; and hence, may be termed as a fibrinspecific agent.
It was perhaps thought that fibrin specificity could be an
absolute necessity for minimising the prevailing risk of
ensuing haemorrhage intimately associated with the
application of thrombolytics ; although the latest fibrin
specific drugs usually give rise to appreciable bleeding in
comparison to the non-specific thrombolytics.

Humulin : Humulin

Humulin : Humulin is the branded product of the famous pharmaceutical


manufacturer, Lilly, containing human-insulin and its host of variants, being
produced by it in different countries across the globe.
Description of Insulin :

Insulin is a pancreatic-hormone essentially involved in the regulation of bloodglucose concentrations and also having a specific role in the protein and lipid
metabolism.

In usual practice, the human, porcine, bovine or mixed porcine-bovine insulin is


administered to such patients having insulin-dependent diabetes mellitus in the
management and control of their blood-glucose concentrations.
It may also be used necessarily in certain non-insulin-dependent diabetics. Insulin is
also an essential component of the emergency management and control of diabetic
ketoacidosis.

Insulin is a hormone produced by the -cells of the islets of Langerthans of


the pancreas and essentially comprise of two separate chains of amino acids,
the A and B chains, joined together by two disulphide bridges.

Human insulin
Human insulin is a dimer comprising one chain of 21 amino
acid (A chain) and the other 30 amino acids (B chain).
Both the chains A and B are derived from a single polypeptide
chain, and are held together by two disulphide bridges.
Generally, the insulin gene codes for preproinsulin, a
polypeptide containing the A and B polypeptides of the active
insulin, and a connecting polypeptide that is absent from
mature insulin.
Insulin is processed from preproinsulin via proinsulin by
enzymatic cleavage of the connecting polypeptide from the A
and B chains.

Production of human insulin


One of the processes developed
by Eli lilly in collaboration with
Genetech involves the followin
steps:
The genes (=DNA sequences) for
chains A and B of insulin were
synthesized separately. Each
gene contains methionine codon
at 5 end and stop codon at 3
end.
These gene were integrated
separately with the lacZ gene
(encoding -galactosidase) of
two separate pBR322 plasmids.

These plasmids were transformed into E. coli strains.


The transformed bacteria thus produces the fusion protein of
A chain and B chain separately.
The A and B chains were separated by a methionine residue
from the -galactosidase sequence encoded by lacZ.
Therefore, the insulin sequences were separated from the galactosidase sequences by treating the fusion proteins with
cyanogen bromide.

The purified chains A and B were then attached to each other


by disulphide bonds to produce insulin.

This method however turned out to be an inefficient


reaction.
Therefore, a gene representing B, C and A Chains was
synthesized and expressed in E. coli ; in this case, the
intervening chain is removed proteolytically
following spontaneous folding of the pro insulin
molecule.

Hepatitis B [Recombivax HM (Merck) A Hepatitis B Vaccine]

The Recombivax HB (Merck), a hepatitis B vaccine, is one of the most


recent and significant developments in the field of recombinant DNA
technology, that essentially comprise of highly specific antibodies which
act like magic bullets.
It has been duly observed that hepatitis tends to cause a severe acute
infection and may ultimately lead to chronic infection and permanent
liver damage. It is essentially caused by hepatitis B virus (HBV) ; and
recognized as an enveloped and double-stranded DNA virus.
It has been adequately revealed through meticulous studies that
individuals who are at the most vulnerable and greatest risk for infection
include : IV-drug abusers (e.g., morphine/heroin addicts) ; homosexual
men ; HBV-infected mothers ; and above all the health care workers.
Various steps being followed in a sequential manner with regard to the
production of a genetically engineered vaccine e.g., Hepatitis B Vaccine.

Step 1 : Genetic material (DNA) is extracted from the ensuing hepatitis virus. At
this stage the surface proteins essentially provoke an immune response.
Step 2: The individual genes are adequately analyzed and identified.
Step 3 : The specific gene which categorically directs production of surface
protein is located carefully.
Step 4 : In this most critical steps the gene is removed from the viral DNA and
inserted into the plasmid carefully.
Step 5: The plasmids are meticulously inserted into the corresponding yeast cells.
Step 6 : Yeast is allowed to grow via fermentation. In this manner the cells
reproduce and generate more quantum of surface protein.
Step 7 : After a duration of 48 hours the corresponding yeast cells are ruptured to
free the ensuing surface protein. The resulting mixture is duly processed so as to
extract the purify
the surface protein.
Step 8 : A large amount of surface protein particles, in its purest form, are
obtained which
ultimately provoke an immune response effectively.
Step 9 : The resulting surface proteins are adequately mixed with appropriate
preservations together with other ingredients to obtain the vaccine.

Humatrope [Growth Hormone]


Growth hormone is an anabolic hormone secreted by
the anterior pituitary which stimulates tissue growth
and anabolism. It is found to affect fat, carbohydrate
and mineral metabolism.
Humatrope (somatropin):
It is a polypeptide hormone of rDNA origin.
Manufactured by Eli Lilly and Company, it is used to
stimulate linear growth in pediatric patients who lack
adequate normal human growth hormone. It has 191
amino acid residues and a molecular weight of
22,125 daltons.

Its amino acid sequence is identical to that of human growth


hormone of pituitary origin (anterior lobe). Humatrope is
synthesized in a strain of E. coli modified by the addition of a
gene for human growth hormone.

Other human growth hormone produced from


rDNAs
include; Omnitrope (Sandoz), Nutropin, Norditropin, Genotrop
in (Pfizer).
Units : One Ampoule of the First International Standard (1987)
: 4.4 units of the human growth hormone (somatropin) are
contained in 1.75 mg of freeze-dried purified human growth
hormone, with 20 mg of glycine, 2 mg of mannitol, 2 mg of
lactose, and 2 mg of sodium bicarbonate.

Pharmacokinetics :
Somatropin is well-absorbed after subcutaneous or IM
injection.
After IV injection it has a half-life of about 20-30 minutes ;
however, after subcutaneous or IM administration the
prevailing serum concentrations usually decline having a halflife of 3-5 hours, on account of the relatively more prolonged
release from the site of injection. It is found to be metabolised
in the liver and excreted in bile.

Uses and Mechanism of Action :


Somatropin is a synthetic human growth hormone ; and
Somatrem is its corresponding methionyl analogue. The drug
promotes the growth of muscular, skeletal, and other tissues,
stimulates protein anabolism ; and also affects fat and mineral
metabolism.
The hormone exhibits a diabetogenic action upon the
carbohydrate metabolism specifically.
The secretion is observed to be pulsatile and solely depends
upon the neural and hormonal influences, such as : (a)
hypothalamic release-inhibiting hormone e.g., somatostatin,
and (b) hypothalamic releasing hormone e.g., somatorelin. In
fact, there are certain physical and physiological factors that
largely influence an enhanced secretion of the growth
hormone, such as : sleep, emotional stress, and
hypoglycaemia.