Transposons

Dr Derakhshandeh
 Mobile Genetic Elements

 Transposons or Transposable elements

(TEs)

 move around the genome

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 Transposable elements in
prokaryotes

 Insertion sequence (IS) elements

 Transposons (Tn)

 Bacteriophage Mu

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 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’s glactose 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)
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Integration of IS element in
chromosomal DNA

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 Three different mechanisms
for transposition
 Conservative transposition

 Replicative transposition

 Retrotransposition

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 Three different mechanisms
for transposition
 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)
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Conservative transposition

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Replicative transposition

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Retrotransposition

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 common feature of mobile elements

 Generation of short direct repeats flanking the

newly inserted element

 This results for a staggered cut being made in

the DNA strands at the site of insertion

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 Transposons (Tn)

 Similar to IS elements but are more

complex structurally and carry additional
genes

 2 types of transposons:

 Composite transposons

 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

 Tetracycline resistance is carried by a

transposable element
 The transposon is a composite transposon,
composed of IS-elements flanking an included
sequence, in this case containing an antibiotic
resistance gene
 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 17
transposition of the entire transposon
Noncomposite
transposons

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 Noncomposite transposons
(Tn)
 Carry genes (e.g., a gene for antibiotic
resistance)

 Ends are non-IS element repeated sequences

 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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 Examples of DNA-intermediate
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) 20
 Methods for Generation of
Mutant Populations

 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:
 1. Insertional mutagenesis
 2. Deletion mutagenesis
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 Two main methods

 1. Transposon insertion

 2. T-DNA insertion

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 Transposon mutagenesis

 Transposable elements or transposons

 sections of DNA (sequence elements)

 move, or transpose, from one site in the

genome to another

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All transposable elements fall into one of
the following two classes

 1. DNA elements

 2. Retroelements

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 DNA elements
 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

 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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 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
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Retrotransposon transposition

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 Retorviruses

 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?
 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

 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. 2001. Proc Natl Acad Sci U S A. 2001 24;98(9):5110-5

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 T-DNA insertion mutagenesis

 T-DNA is a segment of the tumor-

inducing (Ti) plasmid of Agrobacterium

 delimited by short imperfect repeat

border sequences

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 T-DNA transfer from
Agrobacterium to plant cell

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 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

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bacteriophage Mu

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 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
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 Drosophila transposons
 ~15% of Drosophila genome thought to be mobile
 2 different classes:

 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
41 wa)
 ITR(17bp) ITR(17bp)
DTR DTR

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 P elements

 Hybrid dysgenesis, defects arise from crossing of

specific Drosophila strains

 Occurs when haploid genome of male (P strain)

possesses ~40 P elements/genome

 P elements vary in length from 500-2,900 bp

 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 43
repressor in cytoplasm)
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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:
 (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 50
 Ac/Ds Transposon tagging
system

 Advantages: Efficient and cost-effective

method to generate a large mutant population

 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

 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)

 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)

 short (about 100-400 bp)

 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 XIIIB gene

 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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