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Transposons
Dr Derakhshandeh
Mobile Genetic Elements
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Transposons or Transposable elements
(TEs)
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move around the genome
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Transposable elements in
prokaryotes
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Insertion sequence (IS) elements
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Transposons (Tn)
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Bacteriophage Mu
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Insertion sequence (IS)
elements
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Simplest type of transposable element found in
bacterial chromosomes and plasmids
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Encode only genes for mobilization and insertion
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Range in size from 768 bp to 5 kb
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IS1 first identified in E. coli’s glactose operon is 768
bp long and is present with 4-19 copies in the E.
coli chromosome
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Ends of all known IS elements show inverted
terminal repeats (ITRs)
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Integration of IS element in
chromosomal DNA
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Three different mechanisms
for transposition
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Conservative transposition
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Replicative transposition
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Retrotransposition
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Conservative transposition:
The element itself moves from the
donor site into the target site
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Replicative transposition:
The element moves a copy of itself to
a new site via a DNA intermediate
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Retrotransposition:
The element makes an RNA copy of itself
which is reversed-transcribed into a DNA
copy which is then inserted (cDNA)
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common feature of mobile elements
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Generation of short direct repeats flanking the
newly inserted element
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This results for a staggered cut being made in
the DNA strands at the site of insertion
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Examples of DNA-intermediate
mobile elements
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Insertion Sequences (IS) elements in
bacteria
P elements in Drosophila
AC/DS (dissociation) elements in maize
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AC is a full-length autonomous copy
DS is a truncated copy of AC that is nonautonomous, requiring AC in order to transpose
At least seven major classes of DNA
transposons in the human genome (3% of
total genome)
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Methods for Generation of
Mutant Populations
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The most reliable method to ascertain gene
function is to disrupt the gene and determine
the phenotype change in the resulting mutant
individual
Two most popular methods to generate
mutants:
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1. Insertional mutagenesis
2. Deletional mutagenesis
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Two main methods
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1. Transposon insertion
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2. T-DNA insertion
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All transposable elements fall into one of
the following two classes
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1. DNA elements
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2. Retroelements
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DNA elements
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These elements transpose via DNA intermediates
such as:
Ac/Ds and Spm 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
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DNA-Immediate Mobile Genetic Elements
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The Short inverted repeats at the ends of the
element
These inverted repeats act as the substrates
for recombination reactions mediated by the
transposase
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Structure and transposition of a
transposable element
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All transposable elements fall into one of
the following two classes
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1. DNA elements
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2. Retroelements
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Retroelements
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transpose via RNA intermediates
The RNA is copied by reverse
transcriptase into cDNA
the cDNA integrates into the genome
Retroelements are found in all
eukaryotes
such as Tos in rice, copia in animals
Ty1 in yeast
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Retrotransposon transposition
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Retorviruses
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The basic structure is an LTR = long
terminal repeat which flanks three
genes,
A complete retroviruses also contains
three genes:
gag = structural gene for capsid
Pol = reverse transcriptase
env = envelope gene for the virus
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How do we use a transposon
for mutagenesis?
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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
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Transposomics
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EZ::TN Transposomes provide an efficient and reliable method for
generating a library of random gene knockouts in vivo
Gene inactivation and examination of the resulting phenotype will
identify the function of the interrupted genes
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Transposon-Mediated Homologous Recombination
Gene Knockout in Fungi
Hamer et al. Proc Natl Acad Sci U S A. 2001 24;98(9):5110-5
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T-DNA insertion mutagenesis
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T-DNA is a segment of the tumorinducing (Ti) plasmid of Agrobacterium
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delimited by short imperfect repeat
border sequences
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T-DNA transfer from
Agrobacterium to plant cell
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Transposons (Tn)
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Similar to IS elements but are more
complex structurally and carry additional
genes
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2 types of transposons:
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Composite transposons
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Noncomposite transposons
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Composite
transposons
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IS10R is an autonomous element, while IS10L is non-autonomous
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Composite transposons (Tn)
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Carry genes (e.g., a gene for antibiotic
resistance) flanked on both sides by IS
elements
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Tn10 is 9.3 kb and includes 6.5 kb of
central DNA (includes a gene for
tetracycline resistance) and 1.4 kb
inverted IS elements
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IS elements supply transposase and ITR
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recognition signals
Composite Transposons
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Tetracycline resistance is carried by a
Transposable element
The transposon is a composite transposon,
composed of IS-element flanking an included
sequence
IS10R is an autonomous element
while IS10L is non autonomous
Composite transposons probably evolved from IS
elements by the chance location of a pair in close
proximity to one another. Inactivation of one
element by mutation would not harm ability to
transpose and would assure continued
transposition of the entire transposon
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Temperate bacteriophage
Mu (Mu = mutator)
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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
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bacteriophage Mu
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The advantages /
disadvantage of Mu
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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
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Drosophila transposons
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~15% of Drosophila genome thought to be mobile
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2 different classes:
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Copia retrotransposons
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Conserved, 5-100 scattered
copies/genome
Structurally similar to yeast Ty elements
Use RNA and reverse transcriptase
Eye Color in Drosophila (white apricot
40 wa)
DTR
DTR
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P elements
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P elements vary in length from 500-2,900 bp
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possesses ~40 P elements/genome
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Hybrid dysgenesis, defects arise from crossing of
specific Drosophila strains
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Occurs when haploid genome of male (P strain)
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P elements code a repressor, which makes them
stable in the P strain in male (but unstable when
crossed to the wild type female/; female lacks
repressor in cytoplasm)
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Noncomposite
transposons
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Noncomposite transposons
(Tn)
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Carry genes (e.g., a gene for antibiotic
resistance)
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Ends are non-IS element repeated sequences
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Tn3 is 5 kb with 38-bp ITRs and includes 3
genes; bla (-lactamase), tnpA (transposase),
and tnpB (resolvase, which functions in
recombination)
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Ac (activator)/Ds (dissociation) System
discovered by B. McClintock (Noble Prize Winner in 1983)
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Ac/Ds System
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Ac/Ds System
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Schematic Diagram of the Ds Donor Site and
Possible Transposition Events
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Open arrowheads indicate the 5' and 3' ends of th
transposon
The Ds element carries the NPTII gene, which confers
resistance to kanamycin (KanR)
and a modified GUS reporter gene (Sundaresan
et al. 1995 )
Possible transposition events include the following:
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(1) unlinked or loosely linked transposition to the same
chromosome;
(2) transposition to a different chromosome;
(3) closely linked transposition; and
(4) closely linked transposition disrupting theIAAH gene
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Ac/Ds Transposon tagging
system
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Advantages: Efficient and cost-effective
method to generate a large mutant population
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Disadvantages: Secondary transposition
complicates gene identification
And transposon system is not available in
many species
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Transposition
elements in Human
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Mobile Genetic Elements and
Other Families of Repetitive DNA
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The genome is littered with large families of
repetitive sequences
have no apparent function in the cell
Mobile Genetic Elements
Tandemly repeated simple sequence DNAs
 Satellite DNAs
 Short simple repeats (microsatellites)
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LINEs (Long interspersed elements)
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LINEs are one of the most ancient and
successful inventions in eukaryotic genomes
In humans, are about 6 kb long
encode two open reading frames (ORFs)
Most LINE-derived repeats are short, with an
average size of 900 bp - 1,070 bp
The LINE machinery is believed to be
responsible for most reverse transcription in
the genome
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SINEs (Short interspersed
elements)
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short (about 100-400 bp)
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A single monophyletic family of SINEs (ALU)
This family is the only active SINE in the human
genome
The human genome contains three distinct
monophyletic families of SINEs: the active Alu,
and the inactive MIR and Ther2/MIR3
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Identification of a human specific Alu
insertion in the factor XIII gene
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Alu repeats are interspersed repetitive
DNA elements specific to primates that
are present in 500,000 to 1 million
copies
An Alu Insert as the Cause of a Severe
Form of Hemophilia A (factor VIII)
Acta Haematol 2001;106:126–129
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