PROFESSOR JAYASHANKAR TELANGANA STATE

AGRICULTURAL UNIVERSITY
College of Agriculture, Rajendranagar, Hyderabad- 500030

Master’s Seminar (MBB - 591)

Presented To,
Presented by,
Dr. S. Sokka Reddy
Ajay Kumar Chandra
Director
RAM/14-97
Institute of Biotechnology
M.Sc. (Ag) Mol. Biology & Biotechnology
Transposable Elements and their Role in
Plant Genetic Engineering
 What …… we going to discuss today?

• Introduction : Transposable element

• First experiment
• General characteristics of TE
• Transposition and classifications of TEs
• Biological Significance of Transposons
• Effects caused by Transposons
• Transposons:- Role in Plant Genetic Engineering
• Review article and case study
• References ….
 Introduction

• Term was given by Hedges and Jacob (1947)

• Transposon (Transposable element)
A discrete sequence in the genome that are mobile and able to insert
itself at a new location in the genome, without having any sequence
relationship with the target locus.
• Other names viz., Jumping genes, Junk DNA, Selfish DNA,
Molecular parasites, Mobile genetics elements and Controlling
elements.
 What are Transposons?
• Identified in Prokaryotes and all Eukaryotes:
(with exception of parasitic Plasmodium falciparum)
- Animals 3-45%, Fungi 2-20%, Plants 10-80%.
• More abundant in eukaryotic genomes than prokaryotes.
• Transposons make up the major content of eukaryotic genomes.
~50% of genomes of Human, Chimp, Mouse, Ape
~ 10% of several fish species
~ 12 % of C. elegans genome
~75% of Maize genome
~85% of Barley genome
~98% of Iris genome

Iris brevicaulis Iris fulva

Variation in cereal genomes - Transposons & Genome duplications

Rice 450 Mb Sorghum 700 Mb Maize 2,500 Mb

Barley 5,000 Mb Wheat 20,000 Mb Oats ~20,000 Mb

How do organisms live with TEs?

• Most TEs are broken (cannot Tranpose“fossils”)

• Active TEs evolved to insert into “SAFE-HAVENS”

• Host regulates TE movement.

• TEs can provide advantages.

 First experiment
Barbara McClintock (1902-1992)
Cold Spring Harbor Laboratory, NY.

Nobel Prize in Physiology and Medicine (1983)

“Discovery of mobile genetic elements”

Her discovery of Jumping genes, through an

analysis of genetic instability in Maize.

The instability involves chromosome breakage and was found to occur at

sites where transposable elements were located i.e. at C locus of 9th
chromosome.
 Discovery of Ac/ Ds elements
• Genetic marker that McClintock followed is C’ i.e. a dominant inhibitor of
aleurone coloration.
Unexpected corn kernel phenotypes in the offspring of her
corn crosses:
 Dr. McClintock’s Conclusions
There must be two elements to the process:
1). An element that actually caused the mutation
- The dissociator (Ds), located on the short arm of chromosome
9 (where the breaks occurred).

2). An additional element that controlled the activity of the first

element
- The activator (Ac)
Transposition of Ds Element Disrupts Gene Controlling Kernel Color
McClintock’s discovery of transposons
in corn

• c/c = White kernels and C/- = Purple kernels

• Kernal color alleles/traits are “unstable”.
• If reversion of c to C occurs in a cell, cell will produce purple pigment and
a spot.
• Earlier in development reversion occurs, the larger the spot.
• McClintock concluded “c” allele results from a non-autonomous
transposon called “Ds” inserted into the “C” gene.
(Ds = dissassociation)
• Autonomous transposon “Ac” controls “Ds” transposon.
(Ac = activator).
• Ac element is autonomous while Ds element is non-autonomous.
• Ac is 4,563 bp with 11 bp ITRs and 1 transcription unit encoding an
807 amino acid Transposase.
• Ac activates Ds; Ds varies in length and sequence, but possesses same
ITRs as Ac.
• Many Ds elements are deleted or rearranged version of Ac; Ds element
derived from Ac.
• Ac/Ds are developmentally regulated; Ac/Ds transpose only during
chromosome replication and do not leave copies behind.
 General characteristics of TE
• Exist as multiple copies in the
genome.
• Insertion site of element does not have
extensive homology to the transposon.
• Termini are an inverted repeat (ITR).
• Encode “Transposase” that promote
movement .

Features unique to plant transposons:

1). Footprints: when some elements move, leave behind duplicated target
sequence (footprint), which can still affect the gene (only
partial restoration of gene function).
2). Two-element systems: mobility of one element depends on another.
Structure of Ac and its Transposase

Kunse & Weil, 2002

 Transposition of TEs
Transposition: The process by which these sequences are copied and inserted
into a new site in the genome.
Segments of the genome that are capable of moving around to different
locations.
- Transposable elements usually are flanked by repeated sequences .
- often carry “Transposase genes” that confer the transposition ability.

Two basic types of transposition

1). Conservative transposition
- An element leaves one locations and inserts in another .
- Does not change overall copy number.
2). Replicative transposition
- A copy of a transposable element is made and this inserts in a new place.
 Basic classification of TE

“cut-and-paste” “copy-and-paste”

Source:- Lodish et al., Molecular Cell Biology, 7th ed.

Categories of TEs by Transposition
 Basic TE subclasses in plant genomes

/replication
protein + helicase

'rolling circle'

“cut-and-paste”
“copy-and-paste”

Lisch 2013, Nature Rev. Genet.

 Examples: Transposon in plants
Transposon Species

Ac/Ds (Activator/Dissociator)
En/Spm (Enhancer/Suppressor-mutator)
Maize (Zea mays)
Mu (Robertson’s Mutator )
Mpi1

Tam elements (Tam 1, 2 and 3) Snapdragon (Antirrhinum majus)

dTph1, Psl Petunia hybrida
Tag1 Arabidopsis thaliana
Slide Tobacco
Tgm1 Glycine max
Pis1 Pisum sativum
 Biological Significance of Transposons

• They provide a means for genomic change and variation, particularly in

response to stress (McClintock’s "stress" hypothesis).
(1983, Nobel lecture, Science 226:792)
• The movement of a transposable element can generate mutations or
chromosomal rearrangements and thus affect the expression of other
genes.
from - Federoff, N. 1984
• or just "Selfish DNA"?
• No known examples of an element playing a normal role in development.

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 Effects caused by Transposons
• Gene Mutation
- Inactivation of genes by insertion.
- They cause mutation which is used in the production of different colour of grapes,
corn and other fruits.
• Gene Structural Modification After Insertion
- A TE inserts itself into a functional gene, it will probably damage it.
- Insertion into exons, introns, and even into DNA flanking the genes can
destroys or alter the genes activity.
• Gene Movement
• Gene Creation- Pseudo gene Creation
- Fragments in a single TE or fusion of a TE with the regulatory and/or
coding regions of a host gene by transposases.
• Gene Regulation
- Because TEs routinely carry their own regulatory modules that determine
the different expression
- Patterns for TE genes, it is not surprising that TE insertion near a gene
often alters its regulation
 Uses of Transposons

1). As cloning vehicles

Transformation vectors for transferring genes between organisms. Also
drug resistance genes encoded by many transposons are useful in the
development of plasmids as cloning vehicles.

2). Transposons mutagenesis

Use of transposons to increase rate of mutation due to insertional
inactivation
 So are transposons good or bad?
• In the process of inserting into the genome, transposons can
– Interrupt the normal coding of DNA,
– Creating gene mutations with a variety of effects.
– They may turn nearby genes off, preventing their ability to create
protein, or they may turn them on, increasing the amount of protein
made.
• There is evidence that transposons aren’t just “Selfish genes” intent on
replicating themselves or genomic “Junk” that provides no benefit to the
host. They may play a creative role in building new functional parts of the
genome .
• Recent research has shown that transposons may help plants respond and
adapt to environmental stress by regulating other genes.
• In bacteria, transposons often carry genes that impart resistance to
antibiotic substances, helping the bacteria survive.
Pl Tr
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 Transposable element may alter gene
expression

Gene expression may be altered by the presence of a transposable element.

a. An insertion may obliterate the reading frame (phenotypic
effects).
b. A transposable element may contain regulatory elements
(effects on transcription of nearby genes).
c. Transposable elements may contain splice sites (effects on RNA
processing even if the element is in an intron).

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 1). Transposable elements in Antirrhinum majus

Nivea recurrens

Cinnamic acid Chalcone Eriodictyol Dihydroquercetin Cyanidin

28
 Nivea Incolorata Pallida

Cinnamic acid Chalcone Eriodictyol Dihydroquercetin Cyanidin

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 TE affected expression of TF VvmybA1

a). Regulation of antokyan synthesis genes.

(Kobayashi et al. 2004, Science)

 TE affected gene expression

(Yang et al. 2013, PNAS)

Red orange: Ruby – myb TF

(regulation of antokyan genes)
- Activated by TE insertion.
- Cold induced expression
in fruits.

Maize: – inactivation of CCT (photoperiod response) by CACTA-like element

(DNA TE) insertion to promoter.
– Allowed cultivation in temporal climate (long-day
(Butelli et al. 2012,flowering)
Plant Cell)
- Block of branching (TE enhancer  OE of inhibitor)
 Transposon tagging and Gene isolation
• Technique proposed by:- Walbot, 1992
• Loss-of-function mutations.
• Transposon tagging is a gene cloning strategy that relies on the transposon to provide a
DNA “tag” with a known sequence

• The transposon sequence is used to identify DNA sequences adjacent to the

transposable element.

• Tagging an interrupted gene with transposon followed by recognition of its

inactivation on the basis of a phenotype and cloned, using transposon sequence
as a probe.
• Performed by Phenotypic screening, Verification of co-segregation and
isolation of gene.
• Transposable elements are useful tags only if the sequences of the elements are
known.
Schematic of a transposon-tagged
mutation
Transposon tagging and gene cloning
 Tos17 in rice genome

• Tos17 is one kind of copia-like retrotransposons in rice (Hirochika et al.

1996) ,which can duplicate and paste to elsewhere in the genome.
• Tos17 , a rice retrotransposon has been used for large scale mutagenesis.
• Tos 17 is activated only under tissue culture conditions and is not active under
normal conditions.
• By the tissue culture of the rice callus, the transcription of tos17 is activated
and the reverse transcript DNA fragments are integrated into new places in the
genome which creates disruption of genes.

• There are only two copies of Tos 17 under normal conditions in Japanica rice
and 5-30 transposed copies are found in plants regenerated from tissue culture.
Generation of tos17 insertion mutant lines
by tissue culture.
 Transposons used as tags
Transposon Species

Ac/Ds (Activator/Dissociator)
En/Spm(Enhancer/Suppressor-mutator)
Maize (Zea mays)
Mu (Robertson’s Mutator )

Snapdragon (Antirrhinum
Tam elements
majus)
dTph1, Psl Petunia hybrida
Tag1 Arabidopsis thaliana
Slide Tobacco
 List of some genes isolated using transposon tagging in
plants

Gene Gene function Species Transposon

A1 Anthocyanin bisynthesis Mu; En/Spm
A2 Anthocyanin bisynthesis En
BZ2 Anthocyanin pathway Ds; Mu
C1 Regulatory gene En/Spm
Maize
C2 Chalcone synthase En/Spm

Deficiens Regulatory gene A. majus Tam 7

An9 Anthocyanin biosynthesis Petunia dTPh-1
Case study
 Reference
• LITERATURE CITED:
Suzukia M., Miyaharaa T., Tokumotob H., Hakamatsukab T., Godac Y., Ozekia Y.
and Sasaki N. (2014). Transposon-mediated mutation of CYP76AD3 affects
betalain synthesis and produces variegated flowers in four o’clock (Mirabilis
jalapa); Journal of Plant Physiology; 171: 1586–1590.
Nishihara M., Yamada E., Saito M., Fujita K. , Takahashi H. and Nakatsuka T.
(2014). Molecular characterization of mutations in white-flowered torenia
plants ; BMC Plant Biology, 14:86

• BOOKS REFFERED:
MOLECULAR BIOLOGY OF THE GENE: James D. Watson, Tania A. Baker,
Stephan P. Bell and others, 5th edition, Pearson- Benjamin Cummings.
Thanks

Have a nice