TRANSPOSON MEDIATED

MUTAGENESIS

SWETHA
PALB-1236
Jr. MSc ,Plant
biotechnology

content

Transposons
Types of transposon
Type on transposition
Examples of DNA-intermediate mobile elemen
-Transposon mutagenesis
Applications

TRANSPOSONS
(Mobile DNA)
Transposons are segments of DNA that can move
around to different positions in the genome of a
single cell. In the process, they may
causemutations.
These mobile segments of DNA are sometimes
called"jumping genes".

Atransposable element(TE) is aDNA sequence

that can change its relative position (self-transpose)
within the genomeof a singlecell.
The mechanism of transposition can be either "copy
and paste" or "cut and paste".

Transposition can create phenotypically significant

mutationsand alter the cell'sgenome size.
Barbara McClintock's discovery of thesejumping
genes (Nobel prizein 1983).

There are two distinct types:

Class II transposons : These consist of DNA
that moves directly from place to place.
Class I transposons : These are
retrotransposonsthat
first transcribe the DNA into RNA and then
usereverse transcriptaseto make a DNA copy of
the RNA to insert in a new location.

Insertion sequence (IS) elements

Simplest type of transposable element

found in bacterial chromosomes and
plasmids

Encode only genes for mobilization and

insertion

Range in size from 768 bp to 5 kb

IS1 first identified in E. coli galactose

operon is 768 bp long and is present
with 4-19 copies in the E. coli
chromosome

Ends of all known IS elements show

inverted terminal repeats (ITRs)

INTEGRATION OF IS ELEMENT IN
CHROMOSOMAL DNA

Three different mechanisms for

transposition
Conservative transposition
Replicative transposition
Retrotransposition

CONSERVATIVE TRANSPOSITION :The

element itself moves from the donor site into
the target site.

REPLICATIVE TRANSPOSITION :The element

moves a copy of itself to a new site via a DNA
intermediate.

Retrotransposition: The element makes an

RNA copy of itself which is reversed-transcribed
into a DNA copy which is then inserted (cDNA).

Transposons (Tn)
Similar to IS elements but are more
complex structurally and carry
additional genes
2 types of transposons:
Composite transposons
Noncomposite transposons

Examples of DNAintermediate mobile

elements
Insertion Sequences (IS) elements in bacteria
P elements in Drosophila
AC/DS (dissociation) elements in maize
AC is a full-length autonomous copy
DS is a truncated copy of AC that is non-autonomous,
requiring AC in order to transpose
At least seven major classes of DNA transposons in the human
genome (3% of total genome)

There are three main orders of

retrotransposons
Those withlong terminal repeats(LTRs):
encode reverse transcriptase, similar to
retroviruses;
LINEs (Long interspersed elements ):
encode reverse transcriptase, lack LTRs,
transcribed byRNA polymerase II;
SINEs (Short interspersed elements) : do
not code for reverse transcriptase, transcribed
byRNA polymerase III.

Transposon
mutagenesis
Transposable elements or
transposons
sections of DNA (sequence
elements)
move, or transpose, from one site in
the genome to another

Transposon mediated mutation

stratergy

Transposons cause rearrangement of DNA

Homologous recombination between
multiple copies of a transposon cause
rearrangement of host DNA.
Homologous recombination between the
repeats of a transposon may lead to
precise or imprecise excision.

1.Deletion: sequences adjacent to a

transposon are removed
2.Precise excision : The removal of a
transposon plus one copy of the
duplicated sequences.
3.Imprecise excision: Occurs when the
transposon removes itself from the
original insertion site, but leaves
behind some of its sequence.

All transposable elements fall

into one of the following two
classes
1. DNA elements
2. Retroelements

DNA
elements

These elements transpose via DNA intermediates

such as:
Ac/Ds in plants, P elements in animals, Tn in
bacteria
A common feature of DNA elements is the flanking of
the element by short inverted repeat sequences
The enzyme transposase recognizes these
sequences, creates a stem/loop structure
excises the loop from the region of the genome
The excised loop can then be inserted into another
region of the genome

DNA-Immediate Mobile Genetic

Elements

The Short inverted repeats at the ends of the

element.
These inverted repeats act as the substrates for
recombination reactions mediated by the
transposase

Retroelements
Transpose via RNA intermediates
The RNA is copied by reverse transcriptase into
DNA
the DNA integrates into the genome
Retroelements are found in all eukaryotes
such as Tos in rice, copia in animals and Ty1 in
yeast

Retrotransposon transposition

How do we use a transposon for

mutagenesis?
The insertion and excision of transposable
elements
result in changes to the DNA at the
transposition site
The transposition can be identified when a
known DNA sequence or selection markers
are inserted within the elements

Temperate bacteriophage Mu (Mu =

mutator)
37 kb linear DNA with central phage DNA
and unequal lengths of host DNA at each
end
Mu integrates by transposition
replicates when E. coli replicates
During the lysogenic cycle, Mu remains
integrated in E. coli chromosome

The advantages / disadvantage of

Mu
The advantages of the use of Mu are:
it is not normally found in the bacterial genome
therefore there are few problems with homology to
existing sequences in the chromosome; in contrast
to most other transposons
Mu does not need a separate vector system since it
is itself a vector
A wide variety of useful mutants of Mu have been
generated
The disadvantage of Mu:
it is a bacteriophage and therefore can kill the host
cell

Drosophila transposons
~15% of Drosophila genome thought to be
mobile
Copia retrotransposons
Conserved, 5-100 scattered
copies/genome
Structurally similar to yeast Ty
elements
Use RNA and reverse transcriptase
Eye Color in Drosophila (white apricot
wa)

Repicative transposition proceds through a

cointegrates
Cointedrates: A fusion of two original
molecules
-Replication of a strand complex generated a
cointegrate, which is a fusion of the donor and
target replicons.
-The cointegrates has two copies of the
transpose, which is lies between the original
replicons.
--The recombination reaction is catalyzed by a
resolvase coded by the transposon.

Tn A Transposition requires transposase and resolvase

Replicative transposition of TnA requires a

transposase to the cointegrate structure and
resolvase to release to release the two
replicon.
The two stages of TnA transposition are
accomplished by the transposase and the
resolvase , whole genes, trpA and tnpR, are
identified by recessive mutation .The site of
resolution is called res. Resolution occure by
breakage and rejoining bonds without input
of energy .

Non replicative transposition proceds by breakage

and reunion
Non replicative transposition results when a
cross over structure is released by niking .
This insert the transposon into The target
DNA, flanked by the direct repeats of the
target, and the donor is left with in double
standard break.

Controlling elements in maize cause

breakage and rearrangements

Transposition in maize was discovered because of the

effect of the chromosome breaks generated by
transposition of controlling elements. The breakage
generate one chromosome that has a centromere and
broken end and one acentric fragment.
The acentric fragment is lost during mitosis , and this
can be detected by a the disappearance of dominant
alleles in a heterozygote .Fusion between the broken
end of the chromosome generate dicentric
chromosomes, which undergo further cycle of breakage
and fusion.

The fusion breakage- bridge cycle is responsible for the

occurencce of somatic varigation .Clonal analysis
identifies a group of cells descended from a single
ancestor in which a transposition mediated events
altered the phenotype. Timing of the events during
development is indicated by the number of cells ; tissue
specificity of the event may be indicated by the location of
the cells

A break at a controlling element causes loss of an acentric

fragments ; if the fragments carries the dominant marker
of a heterozygote ,its loss change the phenotype.

Applications
Transposable elements as a genetic tool
The first TE was discovered in the plant
maize(Zea mays, corn species), and is
nameddissociator(Ds).
Likewise, the first TE to be molecularly
isolated was from a plant (Snapdragon).
TEs have been an especially useful tool in
plant molecular biology. Researchers use
them as a means of mutagenesis.

 The insertion of a TE into a gene can disrupt

that gene's function in a reversible manner, in a
process calledinsertional mutagenesis;
transposase-mediated excision of the DNA
transposon restores gene function. This produces
plants in which neighboring cells have different
genotypes. TEs are also a widely used tool for
mutagenesis of most experimentally tractable
organisms.
TheSleeping Beauty transposon systemhas
been used extensively as an insertional tag for
identifying cancer genes.
The Tc1/mariner-class of TEsSleeping Beauty
transposon system, awarded as the
Molecule of the Year2009 is active in mammalian

Case study
Transposon-mediated Insertional
Mutagenesis in Gene Discovery and
Cancer
Jun Kong

During experiment they tried to incorporate

insertional mutagenesis tools for in vivo
studies to generate mouse models of various
cancer types. These experimental strategies
are novel in that mutagenesis is taking place
under a specific genetic background to
trigger disease. These experiments are
designed to keep a right balance between
the oncogenic fusion protein and
mutagenesis rate, so that mutagenesis rate
is not too high to override the oncogenic
effect of the fusion protein, and is also not
too low to lose the efficiency of
transformation.

Similarly, another group showed that activation

of the transposon in the gastrointestinal tract
epithelium could give
rise to neoplasia, adenomas and
adenocarcinomas (188).
These recent applications based on transposon
mutagenesis highlighted the potential of this
strategy to identify tissue-specific cancer genes
that are relevant for human cancer.

REFERENCE
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310318, 2009.
R. N. Chopra, S. L. Nayar, and I. C. Chopra,Glossary of Indian
Medicinal Plants, Council for Scientific and Industrial Research,
Government of India, New Delhi, India, 1992.
G. Pandey,Dravyaguna Vijnana, Part III Reprint, Chowkhamba
Krishnadas Academy, Varanasi, India, 2004.
Kasarskis, A., Manova, K. and Anderson, K.V. (1998) phenotype
based screen for
embryonic lethal mutations in the mouse. Proceedings of the
National Academy of Sciences of the United States of America, 95,
74857490.

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