ON

ENZYMES USED IN GENETIC ENGINEERING

SUBJECT:- CONCEPTS IN BIOTECHNOLOGY

SUBJECT CODE:-BTC-012

SUBMITTED TO:- SUBMITTED BY:-

Mr. HARSH KUMAR ASHUTOSH

(Lect. In BIOTECHNOLOGY) B.Tech(Bio Tech)- 3rd sem

Section- B

ROLL NO. R109A30

Reg. NO.1040070139

LOVELY PROFESSIONAL UNIVERSITY

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 PREFACE

Genetic engineering is a new technology that combines genes from totally unrelated
species, in combinations not possible using conventional breeding methods. Genes from
an animal, say, a fish, can be put into a plant, a strawberry for instance.. So different type
of enzymes are used in genetic engineering

In fact this is an actual example of an attempt to "improve" strawberry plants. The fish
gene is supposed to make the strawberries more resistant to frost by causing the
strawberry plant to produce a form of antifreeze which the fish normally produces to
endure cold ocean conditions.

The primary goal of this term parer has been to provide useful information about genetic
engineering in a simple and illustrative language . The organization of the material Is
largely traditional and the order in which the content arranged, is a matter of personal
choice. I hope you will enjoy reading.

 Thank you

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 ACKNOWLEDGEMENT

I would like to express my gratitude to all those who gave me the possibility to complete
this term paper. I want to thank the Department of BIOTECHNOLOGY of LOVELY
PROFESSIONAL UNIVERSITY for giving me permission to commence this Term
paper, to do the necessary research work and to use departmental data. I have furthermore
to thank to Mr. HARSH KUMAR , Lect. In CONCEPTS OF BIOTECHNOLOGY who
gave and confirmed this permission and encouraged me to go ahead with my term paper.

I am deeply indebted to my supervisor Lect. HARSH SIR whose help, stimulating

suggestions and encouragement me in all the time of research for and writing of this term
paper.

 Thank you

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 CONTENTS

 1.INTRODUCTION

 2.INTRODUCTION TO GENETIC ENGINEERING

 3. ENZYMES IN GENETIC ENGINEERING

 4.RESTRICTION ENZYME AND THEIR MODE OF ACTION

 5.DNA LIGASE

 6.PROPERTIES OF RESTRICTION ENZYME

 7.EXAMPLE OF RESTRICTION ENZYME

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 INTRODUCTION

Biotechnology has to do with the manipulation of organisms to get useful products. One
of the basis of biotechnology is genetic transformation. Genetic transformation
occurs when DNA is taken in and expressed by a cell from a living organism. Hence
different type of enzymes are required for the same,like restriction enzyme, ligase.

In this term paper we will discuss about the different enzymes used in genetic
engineering, their mode of action along with their structure.Different type of
enzymes work in different manner to the DNA of host.

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Genetic Engineering

Who would have thought that a tomato could possess characteristics of a fish? What
about a plant possessing characteristics of a firefly; or a pig with human traits? These
things may sound like science experiments gone wrong, but in truth, these are products of
experiments that went well. The fish-like tomato and others are results of genetic
modification or genetic manipulation, which are more commonly known as genetic
engineering. Genetic engineering is the process of taking genes and segments of DNA
from one species and putting them into another species, thus breaking the species barrier
and artificially modifying the DNA of various species. These changes in DNA result in
an alteration of reproductive and hereditary processes of the organisms since the process
is irreversible and the organism's offspring will also possess this unique DNA (Levine).

Enzymes

Enzymes used in genetic engineering such as restriction endonuclease (biological

scissors), DNA polymerase (for replication of DNA), reverse transcriptase (to make DNA
from RNA) and ligase (to ligate two DNA fragments) are produced by recombinant DNA
technology (by cloning in high copy number plasmids in bacteria). Other enzymes now
routinely produced by recombinant DNA technology are rennin (an enzyme used in
cheese making mentioned above), lipase (cheese making), - amylase (beer making),
bromelain (meat tenderizer, juice clarification), catalase (antioxidant in food), cellulase
(alcohol and glucose production), and protease (detergents).

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Enzymes and Genetic Engineering

1. In genetic engineering, scientists use restriction enzymes to isolate a segment of DNA that
contains a gene of interest, for example, the gene regulating insulin production. 2. A plasmid
extracted from its bacteria and treated with the same restriction enzyme can hybridize with this
fragment’s “sticky” ends of complementary DNA. 3. The hybrid plasmid is reincorporated into
the bacterial cell, where it replicates as part of the cell’s DNA. 4. A large number of daughter
cells can be cultured and their gene products extracted for human use.

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Restriction Enzymes

The first major breakthrough on the road to genetic engineering came with work done on
restriction endonucleases by Herbert Boyer of the University of California at San
Francisco. As defined by Karl Drlica in Understanding DNA and Gene Cloning: A Guide
for the Curious, restriction endonucleases "are a group of enzymes [a special type of
protein] that . . . occur naturally in a large number of different bacterial species, serving
as part of the natural defense mechanism that protects bacterial cells against invasion by
foreign DNA molecules such as those contained in viruses."15

When, for example, a virus attacks a single-celled bacterium, restriction endonucleases

are unleashed and go to work, cutting the invading DNA into small, nonthreatening
pieces. "Crucial to this protective device is the ability of the nuclease to discriminate
between its own DNA and the invading DNA; otherwise the cell would destroy its own
DNA," Drlica says.

This recognition process involves two elements. First there are specific nucleotide
sequences [As and Ts, Cs and Gs] that act as targets for the nuclease. These are called the
restriction sites. Second, there is a protective chemical signal that can be placed by the
cell on all the target sequences that happen to occur in its own DNA. The signal modifies
the DNA and prevents the nuclease from cutting. Invading DNAs, lacking the protective
signal, would be chopped by the nuclease.16

Thus, restriction enzymes have the remarkable ability to recognize specific arrangements
of DNA base pairs—As and Ts, Gs and Cs. They also have the capacity to act like a
molecular scalpel, severing the DNA at exactly the spot where they detect this sequence
of genetic letters. Restriction enzymes are a powerful tool because there are thousands of
them, and each one acts only on a unique arrangement of As and Ts and Cs and Gs.

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The science of genetic engineering originated in the late 1960s and early 1970s with the
discovery of restriction enzymes (Avise, 1998). While investigating how viruses and
rings of deoxyribonucleic acid (DNA) called plasmids infect bacterial cells, recombine,
and reproduce themselves, scientists discovered that bacteria make enzymes, called
restriction enzymes, that cut DNA chains at specific sites. Restriction enzymes recognize
particular stretches of nucleotides arranged in a specific order and cut the DNA in those
regions only. Each restriction enzyme recognizes a different nucleotide sequence. Thus,
restriction enzymes form a molecular tool kit that allows the chromosome to be cut into
various desired lengths, depending on how many different restriction enzymes are used.
Each time a particular restriction enzyme or set of restriction enzymes is used, the DNA
is cut into identical pieces of the same number allowing for precise replication.

Restriction enzymes make it possible to remove a bit of DNA from one organism's
chromosome and to insert it into another organism's chromosome This allows for the
production of new combinations of genes that may not exist in nature. For example, a

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human gene can be inserted into a bacterium or a bacterial gene into a plant. So far,
however, there are limits to this ability. Jurassic Park fantasies notwithstanding, scientists
are currently unable to create a whole new organism starting solely with a test tube full of
nucleotides. They must start with the complete genetic material of an already existing
organism. Thus, genetic engineering allows the addition of only one or a small number of
new characteristics to an organism that remains essentially the same. In addition, only
characteristics that are determined by one or a few genes can be transferred. The current
knowledge of behavioral genetics is not sufficiently advanced to enable scientists to
transfer behavioral traits, such as intelligence, that are a complex mixture of many genes
and ontogenetic factors.

Mode of Action of Restriction Enzymes –

The restriction enzyme binds to the recognition site and checks for the methylation
(presence of methyl group on the DNA at a specific nucleotide).
If there is methylation in the recognition sequence, then, it just falls off the DNA and
does not cut.
If only one strand in the DNA molecule is methylated in the recognition sequence and the
other strand is not methylated, then RE (only type I and type III) will methylate the other
strand at the required position.
The methyl group is taken by the RE from S-adenosyl methionine by using modification
site present in the restriction enzymes.
However, type II restriction enzymes take the help of another enzyme called methylase,
and methylate the DNA. Then RE clears the DNA. If there is no methylation on both the
strands of DNA, then RE cleaves the DNA.
It is only by this methylation mechanism that, RE, although present in bacteria, does not

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cleave the bacterial DNA but cleaves the foreign DNA. But there are some restriction
enzymes which function exactly in reverse mode. They cut the DNA if it is a methylate

These are enzymes that cut DNA at specific sites. They are properly called restriction
endonucleases because they cut the bonds in the middle of the polynucleotide chain.
Some restriction enzymes cut straight across both chains, forming blunt ends, but most
enzymes make a staggered cut in the two strands, forming sticky ends.

The cut ends are “sticky” because they have short stretches of single-stranded DNA with
complementary sequences. These sticky ends will stick (or anneal) to another piece of
DNA by complementary base pairing, but only if they have both been cut with the same
restriction enzyme. Restriction enzymes are highly specific, and will only cut DNA at
specific base sequences, 4-8 base pairs long, called recognition sequences.

Restriction enzymes are produced naturally by bacteria as a defence against viruses (they
“restrict” viral growth), but they are enormously useful in genetic engineering for cutting
DNA at precise places ("molecular scissors"). Short lengths of DNA cut out by restriction
enzymes are called restriction fragments.

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DNA Ligase

This enzyme repairs broken DNA by joining two nucleotides in a DNA strand. It is
commonly used in genetic engineering to do the reverse of a restriction enzyme,
i.e. to join together complementary restriction fragments.

The sticky ends allow two complementary restriction fragments to anneal, but only by
weak hydrogen bonds, which can quite easily be broken, say by gentle heating. The
backbone is still incomplete.

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DNA ligase completes the DNA backbone by forming covalent bonds. Restriction
enzymes and DNA ligase can therefore be used together to join lengths of DNA from
different

Properties of restriction enzymes

Ends
As illustrated, restriction enzymes invariably cut DNA in such a way as to leave a 3'
hydroxyl on one end, and a 5' phosphate on the other.

In addition, most (but not all) enzymes recognize a symmetrical site (see the figures
below).

Another interesting property of restriction enzymes is that while they often recognize a
symmetrical site, they do not always cut at the axis of symmetry. For instance, the
enzyme EcoRI (from Escherichia coli strain RY13 (I guess they didn't want to put all
those letters in the name of the enzyme) and pronounced "echo are one"), recognizes the
site GAATTC and cuts the DNA between the G's and the A's in a manner depicted below.

Note that the cut produces an overhanging 5' single-stranded end of four nucleotides on
each of the two pieces that are newly liberated (this is clearer if you look at the next two
illustrations).

Similarly, the enzyme BglII (from the microorganisms, Bacillus globiggi and universally
and irreverently pronounced BAGEL TWO) recognizes the sequence AGATCT and cuts
between the first A and G residues.Notice that BglII also yields a single-stranded 5' end.

A third enzyme, BamHI (from the bacterium Bacillus amyloliquifaciens) cuts similarly.

In fact, the four nucleotide single-stranded ends are the same for both BglII and BamHI.
Moreover, there are at least two other six cutting enzymes that have been discovered that
leave the same four nucleotide overhang: BclI and XhoII. These overhanging ends are

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very useful because -- under the proper conditions -- they may base pair with each other.
In fact, because of their affinity for one another, they are often called cohesive or sticky
ends. Moreover, if molecules with these ends are treated with the appropriate enzyme --
DNA ligase -- their phosphodiester bonds may be rejoined (ligated). When two ends that
originate from digestion by a single enzyme are ligated, the resulting molecule can be cut
by the same enzyme again. But if the ends of a DNA molecule that originated with a
BamHI cut and a BglII cut are joined together, the new sequence will not be cut with
either enzyme (I'll ask you to explain why this is so in class).

Note that all restriction endonucleases do not generate 5' single-strand overhangs. In fact,
some don't even produce an overhang at all. Several enzymes -- like SacI -- produce 3'
single-stranded sticky ends. And some enzymes -- like PvuII -- cut at the axis of
symmetry, leaving perfectly aligned ends. DNA molecules without overhangs are said to
have blunt ends.

In addition there are restriction enzymes that cleave DNA some distance away from the
sequence that they recognize. For example the enzyme HgaI makes staggered cuts that lie
5 and 10 nucleotides away from a 5 base pair sequence, GACGC. This leaves 5'
overhanging ends, but, in contrast to the enzymes described above, these will be different
almost every time the enzyme cuts .

Examples of restriction enzymes include:

Recognition
Enzyme Source Cut
Sequence
5'GAATTC 5'---G AATTC---3'
EcoRI Escherichia coli
3'CTTAAG 3'---CTTAA G---5'
5'CCWGG 5'--- CCWGG---3'
EcoRII Escherichia coli
3'GGWCC 3'---GGWCC ---5'

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 5'GGATCC 5'---G GATCC---3'
BamHI Bacillus amyloliquefaciens
3'CCTAGG 3'---CCTAG G---5'
5'AAGCTT 5'---A AGCTT---3'
HindIII Haemophilus influenzae
3'TTCGAA 3'---TTCGA A---5'
5'TCGA 5'---T CGA---3'
TaqI Thermus aquaticus
3'AGCT 3'---AGC T---5'
5'---GC GGCCGC---
5'GCGGCCGC 3'
NotI Nocardia otitidis
3'CGCCGGCG 3'---CGCCGG CG---
5'
5'GANTC 5'---G ANTC---3'
HinfI Haemophilus influenzae
3'CTNAG 3'---CTNA G---5'
5'GATC 5'--- GATC---3'
Sau3A Staphylococcus aureus
3'CTAG 3'---CTAG ---5'
5'CAGCTG 5'---CAG CTG---3'
PovII* Proteus vulgaris
3'GTCGAC 3'---GTC GAC---5'
5'CCCGGG 5'---CCC GGG---3'
SmaI* Serratia marcescens
3'GGGCCC 3'---GGG CCC---5'
5'GGCC 5'---GG CC---3'
HaeIII* Haemophilus aegyptius
3'CCGG 3'---CC GG---5'
5'AGCT 5'---AG CT---3'
AluI* Arthrobacter luteus
3'TCGA 3'---TC GA---5'
5'GATATC 5'---GAT ATC---3'
EcoRV* Escherichia coli
3'CTATAG 3'---CTA TAG---5'
5'GGTACC 5'---GGTAC C---3'
KpnI[30] Klebsiella pneumoniae
3'CCATGG 3'---C CATGG---5'
5'CTGCAG 5'---CTGCA G---3'
PstI[30] Providencia stuartii
3'GACGTC 3'---G ACGTC---5'
5'GAGCTC 5'---GAGCT C---3'
SacI[30] Streptomyces achromogenes
3'CTCGAG 3'---C TCGAG---5'
5'GTCGAC 5'---G TCGAC---3'
SalI[30] Streptomyces albus
3'CAGCTG 3'---CAGCT G---5'
5'AGTACT 5'---AGT ACT---3'
ScaI[30] Streptomyces caespitosus
3'TCATGA 3'---TCA TGA---5'
[30]
SphI Streptomyces 5'GCATGC 5'---G CATGC---3'

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 phaeochromogenes 3'CGTACG 3'---CGTAC G---5'
5'AGGCCT 5'---AGG CCT---3'
StuI [31][32] Streptomyces tubercidicus
3'TCCGGA 3'---TCC GGA---5'
5'TCTAGA 5'---T CTAGA---3'
XbaI[30] Xanthomonas badrii
3'AGATCT 3'---AGATC T---5'
* = blunt ends
N = C or G or T or A
W = A or T

The utility of the restriction enzymes

The discovery of these many restriction endonucleases have allowed genetic engineers to
cut pieces of DNA at specific sites and into defined sizes. The result has been that a
scientist can work with a collection of molecules all of the same size and with ends of
known sequence. Restriction enzymes have proved to be valuable analytical and
diagnostic tools as well.

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 References

• TEXT BOOKS:-
 1. Biotechnology-expanding horizons: b.d.singh. Kalyani publishers . 2nd
edition
 2. Molecular biotechnology: s.b.parimrose. Panima publication. 2nd
edition

• INTERNET:-
 Www. Google.com
 www.bionewsonline.com
 www.library.thinkquest.org
 Www.yahoo.com
 www.biotech.about.com/od/whatisbiotechnology
 www.geneticengineering.org

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