GENETICS

&
MOLECULAR BIOLOGY

PART III
Contents:

Introduction to genetic engineering.

Restriction enzymes and Ligases.

Cloning vectors (Plasmids, Cosmids, Phagemids)

Animal Biotechnology- Application with reference to Aquaculture,

Livestalk, and Human health.
Introduction to Genetic engineering

Genetic engineering is a modern biotechnology technique which is used to

modify the genetic makeup of an organism by adding new traits in to it and
there by produce new variety of organisms. The desired trait or gene is cut
from the donor gene and pasted to the carrying vector. Thus formed
recombinant DNA is transferred into the host organism which has to be
modified genetically.

The process of Genetic engineering is made effective by the action of two

enzymes namely, Restriction enzymes and ligases.

Restriction enzymes, called molecular scissors are precisely cut the desired
DNA and its carrying vector, at specific sites , whereas the ligase enzyme,
called Molecular glue paste these DNA fragments into the carrying vector.
Restriction enzymes

Discovery
1952-53: Luria and Human discovered the phenomenon of restriction and modification.
Named as host-induced, or host-controlled, variation.
In 1968 Matthew Meselson and Robert Yuan reported that they discovered an enzyme in
E.coli K-12 –recognize and digest foreign DNA.
They coined the term “ RESTRICTION ENDONUCLEASE ” to refer to this enzyme.

Introduction
Restriction endonucleases RESTRICT viruses.
Viral genome is destroyed upon entry
Restriction endonuclease = Restriction enzymes Endo (inside), nuclease (cuts nucleic acid).
Restriction endonuclease recognizes a short and specific DNA sequence and cuts it from
inside.
The specific DNA sequence is called recognition sequence.
Restriction enzymes
Restriction???
Bacteriophages varied in their ability to grow on different strains of E.coli .
Once growth was achieved on one host strain, the phages could continue to grow happily on
this strain.
However, the phages were now restricted in their ability to grow on other strains.
Nomenclature...
Smith and Nathans (1973) proposed enzyme naming scheme three-letter acronym for each
enzyme derived from the source organism.
First letter from genus
Next two letters represent species
Additional letter or number represent the strain or serotypes
For example. the enzyme Hind II was isolated from Haemophilus influenzae serotype d.
Named for bacterial genus, species, strain, and type
Example: EcoR1
Genus: Escherichia
Species: coli
Strain: R
Order discovered: 1
Restriction enzymes
Restriction-modification (R-M) system
Endonuclease activity: cuts foreign DNA at
the recognition site.
Methyltransferase activity: protects host
DNA from cleavage by the restriction
enzyme.
Methyleate one of the bases in each strand
Restriction enzyme and its cognate
modification system constitute the R-M
system R-M system.

Protection of Self DNA

Bacteria protect their self DNA from
restriction digestion by methylation of its
recognition site. Methylation is adding a
methyl group (CH 3 ) to DNA.
Restriction enzymes
Types of cuts:

Blunt end Cutters

Sticky-end Over-hangers
Restriction enzymes

Classification of restriction enzymes:

Restriction enzymes are classified based on recognition sequence and methylation
pattern. They are of 4 types: Type I Type II Type III Type IV .

Type I
Multi-subunit proteins Function as a single protein complex ( pentameric ).
Contain :
two R (restriction) subunits,
two M ( methylation ) subunits and
one S (specificity) subunit
Cofactors and activators: Mg2+ , AdoMet , ATP
Cleave DNA at random length from recognition site
E.g.: EcoKI , EcoAI , EcoBI , StyLTII , Type I
Restriction enzymes
Type III
Large enzymes Combination restriction-and-modification
Cleave outside of their recognition sequences non- pallindromic .
Require two recognition sequences in opposite orientations within the same DNA molecule.
Cofactors and activators: Mg2+ , AdoMet
No commercial use or availability.
E.g.: EcoP151, EcoPI , HinfII
Type IV
Cleave only modified DNA ( methylated , hydroxymethylated and glucosyl-
hydroxymethylated bases).
Recognition sequences have not been well defined
Cleavage takes place ~30 bp away from one of the sites.
Sequence similarity suggests many such systems in other bacteria and archaea.
e.g.: EcoKMcrBC
Restriction enzymes
Type II
Type II Most useful for gene
analysis and cloning

More than 3500 REs

Recognize 4-8 bp sequences

Need Mg 2+ as cofactor Cut in

close proximity of the recognition
site

Homodimers ATP hydrolysis is not

required

Exhibit great diversity.

E.g.: EcoP151, EcoPI , HinfII

Restriction enzymes

Mechanism of action of RE

The process involves:

1. Non-specific DNA binding

2. Target site location

3. Recognition

4. Catalysis

5. Release.
Restriction enzymes

Applications :
A type of immune system for individual
bacterial strains, protecting them from
infection by foreign DNA.

Restriction enzymes can be used in processes

such as…
 Southern Blotting
 Restriction Fragment Length
Polymorphism
 DNA finger printing
 Molecular diagnostics
 Cloning
 cDNA library construction
DNA Ligases

DNA ligases close nicks in the phosphodiester backbone of DNA.

Biologically, DNA ligases are essential for the joining of Okazaki fragments during
replication, and for completing short-patch DNA synthesis occurring in DNA repair process.

There are two classes of DNA ligases.

The first uses NAD+ as a cofactor and only found in bacteria.

The second uses ATP as a cofactor and found in eukaryotes, viruses and bacteriophages.
The smallest known ATP-dependent DNA ligase is the one from the bacteriophage T7 (at
41KdA).
Eukaryotic DNA ligases may be much larger (human DNA ligase I is > 100KDA) but they all
appear to share some common sequences and probably structural motifs.
DNA Ligases

DNA Ligase Mechanism

The reaction occurs in three stages in all DNA ligases:

1. Formation of a covalent enzyme-AMP intermediate linked to a lysine side-chain in the

enzyme.
2. Transfer of the AMP nucleotide to the 5’ phosphate of the nicked DNA strand.
3. Attack on the AMP-DNA bond by the 3’-OH of the nicked DNA sealing the phosphate
backbone and resealing AMP.

The following figure illustrates the three reaction stages:

Cloning VECTORS

The molecular analysis of DNA has been made possible by the cloning of DNA.
The two molecules that are required for cloning are : the DNA to be cloned and
a cloning vector.

Cloning vector - a DNA molecule that carries foreign DNA into a host cell, replicates inside a
bacterial (or yeast) cell and produces many copies of itself and the foreign DNA.

Three features of all cloning vectors include:

Sequences that permit the propagation of itself in bacteria (or in yeast for YACs).
A cloning site to insert foreign DNA; the most versatile vectors contain a site that can be cut
by many restriction enzymes
A method of selecting for bacteria (or yeast for YACs) containing a vector with foreign DNA;
usually accomplished by selectable markers for drug resistance
Cloning VECTORS

The molecular analysis of DNA has been made possible by the cloning of DNA.
The two molecules that are required for cloning are : the DNA to be cloned and
a cloning vector.

Cloning vector - a DNA molecule that carries foreign DNA into a host cell, replicates inside a
bacterial (or yeast) cell and produces many copies of itself and the foreign DNA.

Three features of all cloning vectors include:

Sequences that permit the propagation of itself in bacteria (or in yeast for YACs).
A cloning site to insert foreign DNA; the most versatile vectors contain a site that can be cut
by many restriction enzymes
A method of selecting for bacteria (or yeast for YACs) containing a vector with foreign DNA;
usually accomplished by selectable markers for drug resistance
Plasmids
Plasmids are small circular DNA molecules that can replicate independently—without being
associated with chromosomal DNA—in bacteria.
All that is required on the plasmid DNA is an origin of replication—the DNA sequence that signals
the host cell DNA polymerase to replicate the DNA molecule.
In addition, plasmid vectors carry genes that confer resistance to antibiotics (e.g., ampicillin
resistance), so bacteria carrying the plasmids can be selected.
Plasmid vectors usually consist of only 2 to 4 kb of DNA, in contrast to the 30 to 45 kb of phage DNA
present in λ vectors, facilitating the analysis of an inserted DNA fragment.
Plasmid vectors allow easier manipulation of cloned DNA sequences than do phage vectors.
To be cloned into a plasmid vector, a fragment of the insert DNA is ligated to an appropriate
restriction site in the vector and the recombinant molecule is used to transform E. coli. Antibiotic-
resistant colonies, which contain plasmid DNA, are selected.
Such plasmid-containing bacteria can then be grown in large quantities and their DNA extracted.
The small circular plasmid DNA molecules, of which there are often hundreds of copies per cell, can
be separated from the bacterial chromosomal DNA; the result is purified plasmid DNA that is
suitable for analysis of the cloned insert.
Plasmids
Cosmids

A cosmid is a bacterial plasmid with the cos sequence from the lambda phage.
COS is a recognition sequence that allows the DNA to be packaged into infectious
phage particles.
This vector has a plasmid origin of replication so the it can be replicated in bacteria.
It also contains a selectable marker, such as resistance to the drug ampicillin, and a
polylinker for cloning in the DNA fragments.
To clone large fragments the plasmid is digested in the polylinker and then 35-45 Kb
fragments are ligated into the ends.
The ligation is performed at high concentrations of vector and insert so that
concatamers (a string of multiple vector and insert fragments) are formed. These are
then packaged into phage particles in an in vitro reaction.
The individual phage particles are then infected into bacteria and the presence of the
cosmid can be selected by plating on the antibiotic plates for the drug resistance
marker.
The large circle of DNA will replicate like a plasmid.
Cosmids
Phagemids

Phagemid vectors are plasmids which have been artificially manipulated so as to contain a
small segment of the genome of a filamentous phage, such as M13, fd or f1.
The selected phage sequences contain all the cis-acting elements required for DNA
replication and assembly into phage particles. They permit successful cloning of inserts
several kilobases long (unlike M13 vectors in which such inserts tend to be unstable).
Following transformation of a suitable E. coli strain with a recombinant phagemid, the
bacterial cells are superinfected with a filamentous helper phage, such as f1, which is
required to provide the coat protein.
Phage particles secreted from the superinfected cells will be a mixture of helper phage and
recombinant phagemids. The mixed single-stranded DNA population can be used directly
for DNA sequencing because the primer for initiating DNA strand synthesis is designed to
bind specifically to a sequence of the phagemid vector adjacent to the cloning site.
Commonly used phagemid vectors include the pEMBL series of plasmids and the
pBluescript family.
This phage gene (M13, fd or f1) have a ability to suppress replication of
other genes than your gene of your interest.
Discussion on Animal Biotechnology applications:

Aquaculture

Livestalk

Human health.
summary

Genetic engineering is a modern biotechnology technique which is made effective by

the action of two enzymes namely, Restriction enzymes and ligases.

Restriction enzymes serve as molecular and the ligase enzyme serve as Molecular glue
.
Engineered DNA molecule is transferred to host cells for replication or to make may
copies/ cloning.
References
1. Bhamrah, H.S. and Kavita Juneja. “Molecular cell Biology”, Anmol publications Pvt.Ltd.
2. Gupta, P.K. (1996) “Genetics” Rastogi Publications.
3. Powar, C.B. (2003) “Genetics” Vol.I & Vol II.
4. Ranga, M.M. “Animal Biotechnology (Agrobios), Published by Agrobios (India).
5. Rastogi, Sharma, V.N. and Anuradha Tandon (1993). “Concepts in Molecular Biology”. Wiley
Eastern Ltd. N. Delhi.
6. Smustad, Simmons, Jenkins (1999). “Principles of Genetics” John Wiley and sons. Inc.
7. Daniel Fairbanks, W.Ralph Anderson. “Genetics, the continuity of life” (1999). Brooks/Cole
Publishing Company, New York.
8. Verma, P.S. & Agarwal V.K. (2004). “Cell Biology, Genetics, Molecular Biology, Evolutionary
Ecology”. S.Chand &Company, Pvt. Ltd. New Delhi.

http://schaechter.asmblog.org/schaechter/2011/05/a-protection-racket.html
http://www.biochem.umd.edu/biochem/kahn/molmachines/replication/DNA%20Ligase.htm
http://amrita.vlab.co.in/?sub=3&brch=186&sim=781&cnt=1
http://www.sumanasinc.com/webcontent/animations/content/plasmidcloning.html
http://www.ncbi.nlm.nih.gov/books/NBK21498/
http://www.ncbi.nlm.nih.gov/books/NBK9950/
http://www.austincc.edu/biocr/1406/labm/ex9a/prelab_9a_2.htm
http://wssp.rutgers.edu/StudentScholars/WSSP04/WSSP-04_pdfs/Lecture_pdf/WSSP-04_Ch2-Plasmids.pdf
https://www.neb.com/products/restriction-endonucleases/restriction-endonucleases/restriction-endonucleases-molecular-cloning-and-beyond
http://2008.igem.org/Team:UC_Berkeley/GatewayPhagemid
http://www.professorcrista.com/files/animations/madigan%20animations/GeneticEngineering.html
http://www.dnai.org/lesson/go/18420/15683
Thank you
Thank you