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GENETIC ENGINEERING/ RECOMBINANT

DNA TECHNOLOGY
August 13, 2013
DNA is a long chain nucleotide having million or billion
nucleotide. It contains hereditary information which is
transferred from one generation to another.
Gene is a small part of DNA. It contains thousands of
nucleotides. It is responsible for hereditary characters.
DNA of different species is different: both quantitatively
and qualitatively. From the information present in DNA,
protein is synthesized. Protein synthesis is regulated and
monitored by information present in DNA. Any mutation/
modification/ change in DNA results in synthesis of
abnormal protein. This is main cause of cancer.
In the synthesis of protein following steps are involved:
Transcription- information is transferred from DNA to
RNA
Translation- information is used to synthesize protein/
information in DNA is decoded to form protein.
Replication- DNA divides to give two daughter cells and
each cell should contain exact information

RECOMBINANT DNA TECHNOLOGY:


We obtain specific genetic information or specific gene
responsible for formation of specific protein. This gene is
placed/inserted in another DNA. This makes the gene
functional. In DNA this gene is transcribed, translated and
replicated and we get the required protein.
E.Coli cant synthesize various proteins like insulin, but if
we insert the gene responsible for insulin formation in it,
then it will produce insulin.
Numerous hormones like somatostatins or proteins like
interferon can be synthesized by using this technology.
There are numerous methods to get specific gene

CLASSICAL
METHOD/
GENOMICCLONING/
FINGERPRINTING OF GENETIC MATERIAL BY USING
PROBE:
DNA has numerous genes. It is fragmented by enzyme
endonucleases. Probes are used to get required
information. Probes are known sequence of nucleotides
and these are radiolabeled or fluorescent. It is easy to
detect/ identify the nucleotides by using probes. Probes
are used to identify unknown sequence of nucleotide.
In the synthesis of protein, certain specific characteristics
are found. As the universal rule, genetic information
always flow unidirectionally i.e. from DNA to RNA and
then to protein. This law is called Central Dogma of
Molecular Genetics. There are some exceptions to this
law. In case of retrovirus (RNA virus), this law is violated.
Information is transferred from RNA to DNA and then to
RNA and protein is synthesized. Enzyme responsible for
this is reverse transcriptase.

We have certain complementary base pair groups for


nucleotides like adenosine bind with thymine and guanine
with cytosine. There is known sequence forming pair with
unknown nucleotide. This can be identified.
This sequence can be detected/ read from genomic
library. The sequence we have is compared with the
information present in the library. Now it is separated
from pool of fragments and inserted in another DNA.
Other methods are not commonly used. Reverse
transcriptase is used to get information from RNA. We get
specific mRNA and we get required information.
Automatic analyzers are also present which identify the
sequencing of amino acids.
Biochemical method is another method.

August 16, 2013


CODONS:
Codons are present in DNA. 64 types of pattern are found
from four bases. These codons are responsible for the
synthesis of thousands of proteins. Synthesis of protein is
arrangement of amino acids. Twenty essential amino
acids are found in nature.
A gene is taken from eukaryotes and is placed in
prokaryotes. Eukaryotes have more developed enzyme
system and it helps in protein synthesis.
INTRONS:
DNA contain numerous codons, some of them are not
expressed in the form of protein. These are called introns,
non-sense DNA or silent DNA.
EXONS:
Codons which are expressed in the form of protein are
called exon or sense strand.
PRE-TRANSLATIONAL PROCESS:
Prokaryotes (bacteria) cant separate these introns. Thus,
gene placed in bacteria must be processed i.e. introns
must be removed before insertion and this is called pretranslational process. If this isnt done then we may get
abnormal protein.
POST-TRANSLATIONAL PROCESS:
In eukaryotes, after translation numerous enzymes are
required, while prokaryotes lack these enzymes. Insulin is
synthesized in the form of two long chains and then
bonded together. Bacteria cant do this bonding. So,
different cultures if bacteria synthesize two different
chains and then in-vitro (outside bacteria) bonding is
done. This process is known as post-translational process.

PROCESS OF GENETIC ENGINEERING:


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It
is
making/synthesi
zing the genome
by
adding
different types of
genes.
INITIATORS:
Initiators are also
added to initiate
the
process.
Genes
responsible for
methionine
synthesis
are
used for this
purpose.
Polymerase
starts
the
process
by
unwinding the

Vector/plasmid is circular part of DNA having genetic


DNA.
material. Bacteriophage viruses are viruses using bacteria
as host for its growth. We can place plasmid in
TERMINATORS:
bacteriophage virus.
Terminators are added to stop the process.
Host must be simple (insertion becomes easy) and most
studied organism (to have idea how it works?).
Enzymes present in bacteria may hydrolyze the
After opening of plasmid (nicking of plasmid) insertion of
desired gene is done.
There must be covalent
Then transcription,
attachment between
Gene inserted in DNA
Getting gene by any
gene and plasmid; also
translation and
must bound covalently
method
between plasmid and
replication will occur
with it.
host
Then this opening is
closed by using enzyme
endonucleases.
These
Transferred to plasmid
Now it is inserted in host
enzymes are also called
Isolated and purified
having hereditary
cell may be bacteria or
ligases.
charcter
yeast
Synthesis of protein is
called culturing. Protein is
synthesized
protein.
So,
a
then separated, isolated,
substance
may
be
added
that
purified
and
then
Plasmid having
Plasmid is opened by
combine with desired protein to
processed.
hereditary character +
endonucleases
desired gene is obtained
form a complex and prevent
hydrolysis of desired protein.
MARKERS:
To check functionality of
Classical example of molecular
organisms certain genes
genetics is somatostatin. Its gene is
are
inserted
whose
And desired gene is
Nick is closed by using
very
simple, well-studied and has
function can be observed.
placed in plasmid
ligases
less number of amino acids.
These genes are known as
markers, for example,
genes for synthesis of antibiotics. If effect of this gene is
August 20, 2013
observed then it is confirmed that protein is also
synthesized.
GENOMIC DNA:
CONSTITUTION OF THE GENOME:

SOMATOSTATIN:
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It is a simple polypeptide containing 14 amino acids. It


regulates numerous hormones. During construction of its
genome many genes are placed along with gene for
somatostatin.
Gene responsible for methionine synthesis is placed to
initiate process. Code of initiator is 5AUG
Termination code is also placed. Codes of terminator are
UAA, UGA or UAG.
Marker gene (Tcr and Apr) are added. Tcr is tetracycline
resistant gene and Apr is Ampicillin resistant gene.
Somatostatin is a small peptide and can be degraded by
bacterial enzyme so gene for -glycoside is used, and
after synthesis it forms complex with -glycoside to
protect the hormone form degradation.
Then after synthesis hormone is purified and separated.
Insulin is formed in two chains and then these genes are
joined. 3 synthetic processes for insulin were reported but
are lengthy. Two different bacteria synthesis two different
chains then these are isolated, purified and joined
together. This joining is called post-translational process
because this process is carried outside bacteria (in-vitro).

Surface of
cell
contain
insulin
receptors
and these
contain
and

chains
linked by

disulphide linkage.
When resistance is developed even if insulin is present it
will not regulate sugar level.
Resistance may develop due to infection or inflammatory
condition.
Increasing dose of insulin may help to maintain the
glucose level.

ORAL HYPOGLYCEMIC AGENTS


August 21, 2013

SULFONYLUREA:
August 21, 2013

INSULIN:
Gene responsible for the synthesis for insulin has 22
exons and 21 introns. These are responsible for encoding.
Insulin receptor also has two chains, which are linked by
disulphide linkage.
In humans, it is product of single polypeptide which
breakdown in two chains. In bacteria, two chains are
formed separately and then joined together.
Insulin regulates number of metabolic processes mainly
maintains blood sugar level. Its deficiency leads to
diabetes. Diabetes is of two types. Type I is due to
absence of insulin and type II is due to insulin receptor
resistance,
In type II, oral hypoglycemic agents can be given but in
type I only insulin is given. Hypoglycemic agents reduce
blood sugar level even when blood sugar level is normal.
Hypoglycemic agents reduce blood sugar level even when
blood sugar is normal. Antihyperglycemic agents only
reduce blood sugar level when t is high. These agents are
safer than hypoglycemic agents.
Insulin receptors are present at the surface of the cell.
Insulin is synthesized by human beings (endogenous
substance).
It is responsible for monitoring or regulating number of
metabolic processes.
Insulin increases membrane transfer of glucose, amino
acid and potassium.
In case of mutation of gene abnormal molecules of insulin
are produced, which lead to deficiency of insulin or may
be absence.

It stimulates the release of insulin. 1st generation include


tolbutamide, chlorpropamide and hexamide. 2nd
generation drugs are glipizide and glibenclamide.

BIGUANIDES:
These are Antihyperglycemic agents. Insulin release is not
affected. These agents increase absorption of glucose in
the peripheral tissues, decrease hepatic output, inhibit
gluconeogenesis and decrease absorption of glucose.
Metformin and phenformin are its example.
Meglitinide is a substituted benzoic acid derivative and its
action is like sulfonylurea i.e. increases pancreatic release
of insulin.

THIAZOLIDINEDIONE DERIVATIVE:
It increases insulin receptor sensitivity and reduces the
resistance. It affect circulatory lipids i.e. disturbs lipid
profile and change pattern of LDL and HDL. It also affects
LFT. Compounds introduced initially were pioglitazone,
rosiglitazone and troglitazone. These agents were
withdrawn from the market.
Pioglitazone has favorable effect on HDL i.e. increases its
level, but it also increases the level of LDL. Sometimes
effect of LDL is more sever and it is not desirable.
August 23, 2013

SULFONYLUREA:
SAR:
Sulfanilamide is a
chemotherapeutic
agent.
It
has
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numerous activities. It was found that it treats typhoid


fever but even then numerous deaths were reported.
Later it was found that it occurs due to fewer nutrients
and less glucose content in the blood. This side effect was
studied and sulfanilamide was used as a lead molecule for
hypoglycemic agents.
Ring of sulfanilamide is essential. Only possible
modification is the single substitution of hydrogen at
SO2NH2 group. More than 1000 substitutions are possible.
R1 is NH2 in sulfanilamide. It is essential for antibacterial
activity. This group was replaced by methyl (CH3) group
and tolbutamide 1st anti-diabetic drug was formed. At R2
position it contains butyl side chain.
In chlorpropamide, R1 is chlorine and R2 is propyl chain
Acetohexamide contains acetyl (COCH3) group at R1 and
cyclohexane at R2 position,
In glibenclamide, both substitutions
have ring structure at R1 substituted
aromatic ring, while at R2 cyclohexane is
present.
Glipizide contains cyclohexane at R2 and
heterocyclic ring at R1.
MODE OF ACTION:
All derivatives are hypoglycemic agents
i.e. enhance insulin secretion from
pancreas.
BIGUANIDES:
Initially, when it was used it showed toxic effect and then
its use was stopped. Later derivatives were formed and
metformin (Glucophage)is still drug of choice.
R1 is phenyl in phenformin
In metformin, R and R1 are methyl
One group is butyl and other is hydrogen in buformin.
These are Antihyperglycemic agents and dont enhance
release of insulin. It acts by other mechanisms like
increasing receptor sensitivity, decreases resistance,
inhibits hepatic output of glucose and decreases
absorption of glucose.
Substituted benzoic acid derivative is structurally related
to sulfonylurea. Action is also same only receptors are
different.

THIAZOLIDINEDIONE:

It disturbs lipid profile thats why not preferred. Some


compounds are still used in combination with caution, as
these compounds are hepatotoxic and increase levels of
ALT.

PROSTAGLANDINS
September 13, 2013
Prostaglandins are inflammatory mediators. 1st time it
was found in prostate gland thats why termed as
prostaglandins. It was thought that only these glands
produce it but later it was found that each and every cell
can form it.
The precursor is prostanoic acid and arachidonic acid.
Eicosanoic acid/ ethanoic acid is composed of 20 carbons.
These have 2 chains with cyclopentane ring. At 1st position
carboxylic group is present and at 20th position methyl
group is present.
Different types of prostaglandins are found and difference
is in the position of the double bonds and number or
position of the functional groups. Two types of functional
groups
are
present
i.e.
ketonic
and
hydroxyl.
Prostaglandin
A: ketonic group is present at 9th position and the double
bond is between 10th and 11th position.
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Prostaglandin B: ketonic group is at 9th position and


unsaturation is between 8th and 12th.
Prostaglandin C: unsaturation is between 11th and 12th
while ketonic group remain at 9th position.
Prostaglandin E:ketonic group is at 9th and hydroxyl is at
11th position
Prostaglandin F: hydroxyl group is at 9th and 11th position

PGE2 causes uterine contraction and used in induction of


labor
Various prostaglandins are involved in GI secretion. They
may inhibit the synthesis of acid and increase the
secretion of mucous and protect theinternal layer or
membrane. It improves renal blood flow. Leukotriene
increases constriction of bronchioles in asthma.

Prostaglandin A indicates that it is stable in acid


Prostaglandin B is stable in base
Prostaglandin E is stable in ether
Prostaglandin F is partitioned in phosphate buffer

EXAMPLE:
Misoprostol is used as an anti-ulcerative agent
Fenprostalene is used to cause abortion
Alprostadil is used to treat erectile dysfunction
Bimatoprost is used to treat ocular hypertension

Prostaglandin E (PGE) can be further classified into PGE1,


PGE2 and PGE3.
During synthesis of prostaglandin pathway involved in the
synthesis of prostacyclin and thromboxane A2 is adopted.
Prostacyclin and thromboxane A2 are components of
clotting system. Prostacyclin is responsible for vessel
dilation and inhibits platelet aggregation. Thromboxane
A2 supports platelet aggregation.
Another pathway adopted while synthesis can be
leukotrienes/ lipoxygenase pathway.
September 17, 2013
Expansion or reduction in cyclopentane chain will
decrease the activity.
Prostanoic acid is also called Eicosanoic acid. It has 20
carbons.
Lipoxygenase pathway and cyclooxygenase pathway are
involved in its synthesis. It is synthesized from arachidonic
acid. From lipoxygenase pathway leukotriene is
synthesized. It is related to allergic reaction. From
cyclooxygenase pathway prostaglandin/ prostacyclin is
formed. It inhibits platelet aggregation and is a weak
vasodilator.
Enzymes which are involved in it are:

Lipoxygenase

Prostaglandin peroxide synthase

Prostaglandin endoperoxide reductase

Prostaglandin endoperoxide E isomerase

Prostaglandin endoperoxide A isomerase

Prostaglandin endoperoxide I isomerase


These are involved mainly in case of cyclooxygenase
pathway.
-OH group is present in Prostacyclin (PGI2) has got
stability and antiplatelet aggregating activity. Modification
of COOH will reduce the activity.
Prostaglandin has anti-inflammatory activity and it is
involved in reduction of pain and fever.
PGI2 inhibits platelet aggregation

ANTIVIRAL AGENTS
August 23, 2013

VIRUS:
Virus is a cellular parasite. It contains one type of nucleic
acid either RNA or DNA, which is surrounded by core of
protein. This core of protein may be surrounded by
another protein called envelope or capsule. It is a cellular
parasite and can survive on host. It uses substrate and
enzymatic pathways of host cells for protein synthesis and
replication.

LIFE CYCLE OF VIRUS:


Virus attaches with outer surface of the host cell. Then it
will be absorbed on the surface. Uncoating will occur after
that i.e. protein surrounding the nucleic acid will be
removed. Nucleic acid of virus will attach with nucleic acid
of host. Then it will use nucleic acid of host for replication.
Different parts of virus will be synthesized at different
sites of host cells. Now these parts will be assembled.
Then we will get virus, this is called budding i.e. release of
mature virus. Now it will affect adjacent cells.
We can target each step of this life cycle by using antiviral
agents.
August 27, 2013

MECHANISM OF ACTIONS:
Antiviral agents act by following mechanisms:

Inhibit uncoating of virus- Uncoating may occur


either after adsorption of virus on the cell surface or after
attachment of virus with the cell.

Inhibition of penetration- virus must penetrate


into the cell whether coated or uncoated for replication

Inhibition of process
o
Fusion with the host cell
o
Transcription
o
Translation
o
Replication

Inhibit assembling of these particles

Inhibit release of mature viruses


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These agents can act by one or more than one of these


mechanisms. If more than one activity is found then it
called broad spectrum antiviral agent.

genital herpes. It is activated by kinases. It inhibits viral


DNA polymerase. Its oral, parenteral and topical dosage
forms are available.

PROBLEMS:

RIBAVIRIN (VIRAZOLE):

Following problems are associated with antiviral agents:

Selectivity- Antiviral agents must only affect virus,


but as virus is attached with the host cell so, it is difficult
to obtain selectivity.

Brad spectrum

Less toxic for host cell- without affecting host cell


must reach to the target

It inhibits protein synthesis. It inhibits specifically RNA


virus like adenovirus, vaccinia virus, influenza virus and
myxoma virus. It is a broad spectrum antiviral
agent. It is activated by viral kinases and inhibits
enzyme
inosine
monophosphate
dehydrogenase and inhibits formation of
guanine.

AMANTADINE/ RIMANTADINE:
Rimantadine is derivative of amantadine. These inhibit
uncoating and penetration of virus. These are mainly
effective against influenza A virus. They have preventive
action and can be used prophylactically. If virus has
already penetrated or infection is there then these are
not effective.

ZIDOVUDINE(RETROVIR):
It is active against retrovirus/ reverse
transcriptase. It is used in the case of AIDS,
herpes and some types of leukemia. It is an
analogue of thymidine.

TRIFLURIDINE/ 2-DEOXY-5-IODOXIDINE:

MISCELLANEOUS COMPOUNDS:

These are antimetabolites and inhibit synthesis of protein.


These are iodinated or fluorinated. 2-deoxy-5-iodoxidine
is structurally related to nucleoside. It is active against
hepatitis B virus. It can be used topically in herpes and
pox virus (0.1% solution). 0.1 and 0.5% ophthalmic
solutions are available. Trifluridine is available in 1%
solution for ophthalmic use.Trifluridine is fluorinated
derivative of pyrimidine.

These are not structurally related to nucleosides. These


are not antimetabolites.
Saquinavir and nevirapine are drugs of this class used to
treat HIV.

ANTIMALARIAL DRUG THERAPY


September 18, 2013
Causative organism is plasmodium parasite. Plasmodiums
are of four types: Plasmodium falciparum, Plasmodium
vivax, Plasmodium ovale and Plasmodium malaria.
In human being, infection is due to falciparum and vivax.
Before 2nd world war few compounds were used as
antimalarial agents, but later parasite become resistant to
these compounds. Now these compounds are not active.
Cinchona alkaloid is the oldest compound and was used
as an antimalarial agent. It is a potent compound. It is
present in highest concentration in bark of the plant.

ACYCLOVIR (ZOVIRAX):
It is active against number of viruses like Herpes simplex
virus, zoster virus and is choice of treatment in the case of

These
agents
are
classified
accordin
g
to
chemical
structure
into
following
classes:

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Aminoquinoline derivatives
Quinidine derivative
4-aminoquinoline derivative
8-aminoquinoline derivative
Biguanide derivative
9-aminoacridine derivative
Few extracted compounds/ isolated

4-AMINOQUINOLINE:
Compounds have quinolone nucleus with
substituted amino group. Derivatives have side
chain in which one hydrogen of amino group is
replaced by the side chain. Side chain has butyl
and diethyl amino group. It is diethylaminobutyl
side chain; its first carbon can be substituted with
methyl and then will be called 1-methyl butyl
diethylamino group. All compounds of 4 and 8aminoquinoline have same side chains.
SAR:
First compound of the series if santoquin. It was
synthesized by Germans. At that time it was considered as
best derivative of 4-aminoquionoline. It has methyl group
at 3rd position. Later, it was found that substitution of
methyl group has decreased the activity.
One of the most important and commonly used derivative
is chloroquine, in which chlorine group is substituted at
7th position of 4-aminochloroquine.
In hydroxychloroquine, OH group is attached with the
chlorine group, and it is more active than chloroquine.
In amodiaquine, cyclic compound is attached
Mefloquine also contain cyclic compound and CF3 group
is also present.
September 24, 2013

PYRIMIDINE:
If we consider structural activity, chlorine group at para
position will increase the activity. If chlorine group is
replaced by methyl group, then activity will be decreased.
Pyrimidine derivatives are effective in exoerythrocytic
stage while, biguanides and proguanine are effective in
pre-erythrocytic stage.
Proguanine is used with atovaquone against the resistant
strain.
HALOFANTRINE:
Halofantrine is recently introduced compound and is used
in combination with biguanides and pyrimidine
derivatives against the resistant strains.

ARTEMISININ:
It is used in diverse traditional medicine. Now, it is
available as one of the important compound and drug of
choice. Two derivatives are available. One is water soluble
and other is lipid soluble. This compound has lactone ring
and oxygen ring. Oxygen ring is cleaved or broken to give
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the superoxide which is responsible for its biological


activity.

LIFE CYCLE:
It is divided into two phases
ASEXUAL PHASE:
It occurs in host, vertebrate or human being. Female
mosquito (anopheles)when bite unaffected person then
plasmodium is transferred through saliva into the blood of
the host. It is then accumulated in the liver and then
transferred to the blood. This stage is known as
erythrocytic stage. When it is in liver it is called
hepatocytic stage. In erythrocytic stage, hemoglobin is
degraded. 30% hemoglobin is degraded. It is used by
parasite for synthesis of their protein. After 3-4 days
symptoms appear like chill, fever, etc.
SEXUAL PHASE:
When a mosquito bites a person protozoid enters the
salivary gland of mosquito and then moves to the
stomach. Gametocytes are oozed and then the cycle
continues
Two derivatives of chloroquine act at extraintestinal
stage. Amebiasis is a stage when protozoa are in intestine.
When it moves and come to liver then these drugs act
here.
We havent got any specific molecule acting
prophylactically.
Halofantrine if mixed with table salt can act
prophylactically.
Attenuated sporozoids or radiated attenuated sporozoids
are also available for prophylactic use.
Pyrimethamine is used in combination to inhibit synthesis
of folic acid.
Atovaquone interferes with electron transport chain. It is
selective inhibitor of electron transport chain in
plasmodium. Superoxide radical is formed and damages
the plasmodium. It is used in combination with
cycloguanil or proguanine against resistant strains. It is
obtained from Chinese plant. Some are water soluble and
some are oil soluble.
Decoding of plasmodium is also done and it is used
experimentally.

October 1, 2013

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