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
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.
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.
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.
Transformed cell
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.
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.
Generalized transduction
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.
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.
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.
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.
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.
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.
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).
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.
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.
EcoRI
BamHI
DpnI
HindIII
BglII
PstI
Sau3AI
KpnI
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.
Cloning cDNA
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.
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.
:::::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).
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.
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.
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.
Humulin : Humulin
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.
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.
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.
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.