TransposoneTransposonePresented by: Salar Bakhtiyari

They are discrete sequence in the genome They are discrete sequence in the genome that are mobile that are mobile they are able to transport themselves to they are able to transport themselves to other location. Other names: other location. Other names:

Jumping genes Jumping genes Selfish DNAsSelfish DNAs Molecular parasitesMolecular parasites Controlling elementsControlling elements

TEs are present in the genome all species of three domains

Transposable Elements

What do we want to know about What do we want to know about mobile genetics elements?mobile genetics elements?

1 – The history of mobile genetic elements1 – The history of mobile genetic elements 2 – The classification of TEs2 – The classification of TEs 3 – The structure of TEs3 – The structure of TEs 4 – The mechanism of transposition 4 – The mechanism of transposition 5 – The effects of TEs on gene and genome5 – The effects of TEs on gene and genome 6 – The use of TEs as molecular tools 6 – The use of TEs as molecular tools

Why study mobile genetic elements?Why study mobile genetic elements?

They are the major forces driving evolutionThey are the major forces driving evolution

They can cause genome rearrangement They can cause genome rearrangement (mutation , deletion and insertion )(mutation , deletion and insertion )

They have wide range of application potentials They have wide range of application potentials

The discovery of mobile genetic The discovery of mobile genetic elementselements

Transposable elements Transposable elements

PhagePhage

Plasmid DNA Plasmid DNA

The discovery of transposable elementsThe discovery of transposable elements

Barbara Mc Clintock discovered TEs in maize (1983)Barbara Mc Clintock discovered TEs in maize (1983)

Her work on chromosome breakage began by Her work on chromosome breakage began by investigating genetic instability (1983)investigating genetic instability (1983)

Observing variegated patterns of pigmentation in Observing variegated patterns of pigmentation in maize plant and kernelsmaize plant and kernels

New kinds of genetic instabilityNew kinds of genetic instability She spent the next tree decades for this genetic She spent the next tree decades for this genetic

elementselements

Controlling elements (1956)Controlling elements (1956)

Barbara Mc Clintock 1902 Barbara Mc Clintock 1902 1980 ( noble in 1984)1980 ( noble in 1984)

Plasmid , phagePlasmid , phage

Cell to cell conjugation Cell to cell conjugation Bactriophage mediated transductionBactriophage mediated transduction Bill Hayes ( 1952 )Bill Hayes ( 1952 ) Ellin Wollman and Francois Jancob , 1961Ellin Wollman and Francois Jancob , 1961 Alan CampbellAlan Campbell

Classification of transposable Classification of transposable elements elements

DNA transposonsDNA transposons RetrotransposonsRetrotransposons

Autonomous and non autonomous elementsAutonomous and non autonomous elements

Both class are subdivided into distinct superfamilies and Both class are subdivided into distinct superfamilies and familiesfamilies

Structure feature , internal organization , the size of target site Structure feature , internal organization , the size of target site duplication , sequence similarities at the DNA and protein duplication , sequence similarities at the DNA and protein levelslevels

Autonomous : they have the ability to excise and transpose Autonomous : they have the ability to excise and transpose non autonomous elements non autonomous elements - They don’t transpose They don’t transpose - They become unstable only when an autonomous member of They become unstable only when an autonomous member of

same family is present elsewhere in the genomesame family is present elsewhere in the genome- They are derived from autonomous elements They are derived from autonomous elements

A family consists of single type of autonomous element A family consists of single type of autonomous element accompanied by many varieties of non autonomous elementsaccompanied by many varieties of non autonomous elements

DNA based elementsDNA based elements

Insertion sequence (IS)Insertion sequence (IS) The simplest (smallest) transposons are The simplest (smallest) transposons are

called IS called IS The IS elements are normal constituents of The IS elements are normal constituents of

bacterial chromosome and plasmidsbacterial chromosome and plasmids Spontaneous mutation of theSpontaneous mutation of the lac lac and and gal gal

operonsoperons They are autonomous units ,each of which They are autonomous units ,each of which

codes only transposase codes only transposase

Structure of ISStructure of IS

Composite transposoneComposite transposone One class of large transposons One class of large transposons

are called are called Composite transposons

They carring the druge marker They carring the druge marker is flanked on either side by is flanked on either side by arms that consist of IS elementsarms that consist of IS elements

IS modules- identical (both functional: Tn9; Tn903) or closely related (differ in functional ability: Tn10; Tn5)

1. A functional IS module can transpose either itself or the entire transposon

Mechanism of transposition

The stugger between the cuts determines the length of the direct repeats.

The target repeat is characteristic of each transposon; reflects the geometry of the cutting enzyme

Direct repeats are generated by introduction of staggered cuts whose protruding ends are linked to the transposon.

Mechanism of transposition1- Replicative transpositonReplicative transpositon

1. Replicative : a) Transposon is duplicated; a copy of the original element is

made at a recipient site(TnA); donor keeps original copy

b) Transposition- an increase in the number of Tn copies

c) ENZs: transposase (acts on the ends of original Tn) and resolvase (acts on the duplicated copies)

Mechanism of transposition2 -Nonreplicative

Nonreplicative : Transposon moves from one site to another and is conserved; breaks in

donor repaired b) IS and Tn10 and Tn5 use this mechanism; no Tn copy increase c) ENZs: only transposase

The first stages of Both replicative and non-replicative transpositio are semilar

IS elements, prokaryotic eukaryotic

transposons, and bacteriophage Mu.

1. Synapsis stage- two ends of transposon are brought together

3.. Nicked ends joine crosswise;covalent connection between the transposon the target

2. Transposon nicked at both ends; target nicked at both strands

Donor cutDonor cut

cuts in trans

transfers in trans

22 bp

Mu integrates by nonreplicative transposition; during lytic cycle- number of copies amplified by replicative transposition

- MuA binds to ends as tetramer forming a synapsis.- MuA subunits act in trans to cut next to R1 and L1 (coordinately; two active sites to manipulate DNA).

- MuA acts in trans to cut the target site DNA and mediate in trans strand transfer

The chemistry of Donor and target cutThe chemistry of Donor and target cut

The The 3’-ends ends groups released from flanking groups released from flanking DNA by donor cut reactionDNA by donor cut reactionThey are nuclophile that attack phosphodiester They are nuclophile that attack phosphodiester bonds in target DNAbonds in target DNA

Cutting of both endsCutting of both ends

33 ‘‘ OH

33 ‘‘ OH

33 ‘‘ OH

33 ‘‘ OH

Cutting of Cutting of 33 ‘‘ end only end only

The product of these reaction is The product of these reaction is strand transfer complex strand transfer complex

In strand transfer complex transposon is connected to the target site through one strand at each end

Next step differs and determines the type of transposition:

Strand transfer complex can be target for replication (replicative transposition) or for repair (nonreplicative transposition; breakage & reunion)

transposon target

Strand transfer complex

Molecular mechanism of transposition (I)

Replicative transpositio

n

Replicative transposition proceeds through a cointegrate.

Transposition may fuse a donor and recipient replicon into a cointegrate. Resolution releases two replicons-each has copy of the transposon

Replicative transposition

Ligation to target ends

3. 3’-ends prime replicationThe crossover structure contains a single stranded region at each of the staggered ends= pseudoreplication forks that provide template for DNA synthesis

Donor and target cut

cointegrate.

Non-replicative

Replicative

additional nicking

common structure

Breakage & reunion

Retrotransposon ( retroposons )Retrotransposon ( retroposons )

Use of an RNA Use of an RNA IntermediateIntermediate element is transcribedelement is transcribed reverse transcriptase reverse transcriptase

produces a double-produces a double-stranded DNA copy for stranded DNA copy for insertion at another siteinsertion at another site

they as other elements they as other elements generating short direct generating short direct repeat repeat

Types of RetrotransposonsTypes of Retrotransposons

1 – viral superfamily 1 – viral superfamily (autonomousretrotransposon)(autonomousretrotransposon) retrovirusretrovirus LTR- retrotransposon LTR- retrotransposon LINESLINES

2 – nonviral superfamily 2 – nonviral superfamily (non autonomous retransposons) (non autonomous retransposons)

SINES SINES

non LTR- retrotransposonnon LTR- retrotransposon

retrovirusretrovirus

RNA

reverstranscriptase

Liner DNA

Integration

provirus

Transcription

RNA

LTR - retrotrasposonLTR - retrotrasposonpol

Reverse transcriptase (RT)Integrase (IN)

Ribonuclease H (RH)

gag

env

?

mechanism of transposition

Integrase acts on both the retrotransposon line DNA and target DNA

The integrase bring the ends of the linear DNA together-Generate 2 base recessed 3’3’ -ends and staggered end in target DNA

3’-ends

5’-ends

Non – LTR retrovirusNon – LTR retrovirus LINES = long interspersed elementsLINES = long interspersed elements SINES = short interspersed elementsSINES = short interspersed elements don’t terminate in the LTRs don’t terminate in the LTRs they are significant part of relatively short sequence they are significant part of relatively short sequence

of mammalian genomes .of mammalian genomes .

Effect of transposabli elements on gene and genome Effect of transposabli elements on gene and genome

TEs cause a varity of change in the genome of their TEs cause a varity of change in the genome of their hosts hosts

this ability to induce mutation depend on their of this ability to induce mutation depend on their of capability of transposing capability of transposing

some arrangement can be beneficial they can some arrangement can be beneficial they can advantageous for adaptation to new environment advantageous for adaptation to new environment

play important role in evolution .play important role in evolution .

they have the ability to rearrange genomic they have the ability to rearrange genomic information in several ways information in several ways

1 – Modification of gene expression 1 – Modification of gene expression 2 – Alternation gene sequence 2 – Alternation gene sequence 3 – Chromosomal structural changes 3 – Chromosomal structural changes

Modification of gene expression Modification of gene expression

insertion of a TE within or adjacent to a gene insertion of a TE within or adjacent to a gene the element blocks or alters the pattern of the element blocks or alters the pattern of

transcription . transcription . iinsertion ofnsertion of Fot1Fot1 in a intron of in a intron of niadniad ((F . oxysporum F . oxysporum )) different mutant transcripts all were shorter different mutant transcripts all were shorter They result from: They result from:

- presence of termination signal - presence of termination signal

- presence of an alternative promotor- presence of an alternative promotor

Alternation gene sequence Alternation gene sequence

cut and pate mechanism often produce variation cut and pate mechanism often produce variation when they excise .when they excise .

the excision process may result in addition of a few the excision process may result in addition of a few base pair ( footprint ) at donor site .base pair ( footprint ) at donor site .

these footprint cause diversification of nucleotide these footprint cause diversification of nucleotide sequence and new functional allelessequence and new functional alleles

Example :Example :Fot1Fot1 and and Impala Impala generally leave 4 bp – ( 108 ) or 5 – ( 63 ) foot generally leave 4 bp – ( 108 ) or 5 – ( 63 ) foot printsprints

excision excision of the of the Asco - 1Asco - 1 transposon in transposon in A .immersusA .immersus Deletions of a a few to up to 1713 nucleotide in Deletions of a a few to up to 1713 nucleotide in b2 b2 genegene larger deletion led to larger deletion led to variety of phenotypesvariety of phenotypes in spore coloration in spore coloration

Chromosomal structural changesChromosomal structural changes TEs can produce a series of genome rearrangment TEs can produce a series of genome rearrangment

through ectopic recombination through ectopic recombination deletion , duplication , inversion and translucation deletion , duplication , inversion and translucation

mediate by TEs ( Drosophila , Yeast , human )mediate by TEs ( Drosophila , Yeast , human ) karyoptypic variation in natural isolate in fungai karyoptypic variation in natural isolate in fungai high level of chromosome – length polymorphism high level of chromosome – length polymorphism

((Magnoporthe griseaMagnoporthe grisea , , F.F. oxysporumoxysporum))

translocation translocation tox1 tox1 of of Cochliobolus heterostrophusCochliobolus heterostrophus appearance of new virulence alleles in appearance of new virulence alleles in M . grisea M . grisea

Use as strain specific diagnostic toolsUse as strain specific diagnostic tools

TEs are often restricted to specific strains TEs are often restricted to specific strains identify specific pathogen in plant pathology identify specific pathogen in plant pathology Fot1 Fot1 ( ( F. oxysporumF. oxysporum f sp. f sp. albedians albedians ) provide ) provide

PCR targets PCR targets a sensitive detection thechnique to prevent the a sensitive detection thechnique to prevent the

introduction of pathogenic form introduction of pathogenic form

- - race of race of F. oxysporumF. oxysporum responsible of carnation wilt responsible of carnation wilt

- date palm pathogen - date palm pathogen

Use of TEs as molecular toolsUse of TEs as molecular tools

Use of TEs as molecular toolsUse of TEs as molecular tools

MGR 586 MGR 586 (( Magneporthe grisea Magneporthe grisea ) ) oryza : 30 – 50 wheat and other ( 1 – 2 )oryza : 30 – 50 wheat and other ( 1 – 2 ) they have used to distinguish genetically they have used to distinguish genetically

divergent population divergent population fingerprinting of isolates pathogenic to fingerprinting of isolates pathogenic to

oil palm tree. ( oil palm tree. ( F. oxysporumF. oxysporum, , palmpalm))

Tools for the analysis of population structureTools for the analysis of population structure

Gene tagging with transposable elementsGene tagging with transposable elements

arise mutant phenotype

Disrupt target geneDisrupt target gene

Use of TEs as molecular toolsUse of TEs as molecular tools

jumping into coding jumping into coding regionregion

Target gene can easily determined by PCR

methods

Target gene can easily determined by PCR

methods

Thanks for attentionThanks for attention