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Transposable Element(Transposon)

Transposable elements in eukaryotes :

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

Nobel Prize in Physiology and Medicine 1983

“for her discovery of mobil genetic elements”

• Studied transposable elements in corn (Zea mays) 1940s-1950s(formerly identified as mutator genes by Marcus Rhoades 1930s)

Transposable element : genetic elements of a chromosome that havethe capacity to mobilize and move from one location to another inthe genome.

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Normal and ubiquitous components of prokaryote and eukaryotegenomes.

Nonhomologous recombination : transposable elements insert intoDNA that has no sequence homology with the transposon.

Transposable elements cause genetics changes and make importantcontributions to the evolution of genomes:

•Insert into genes.

•Insert into regulatory sequences; modify gene expression.

•Produce chromosomal mutations.

Transposable Elementsin Prokaryotes

1. Insertion sequence (IS)

2. Transposons (Tn)

3. 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.

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 ispresent with 4-19 copies in the E. coli chromosome.

Ends of all known IS elements show inverted terminal repeats(ITRs).

Transposition of insertion sequence (IS) elements :

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Original copy remains in place; new copy inserts randomly.

IS element uses host replication enzymes for replication.

Transposition requires transposase , coded by the IS element.

Transposition initiates when transposase recognizes ITRs .

Site of integration = target site .

Staggered cuts are made in DNA at target site, IS element inserts,DNA polymerase and ligase fill the gaps.

Small direct repeats (~5 bp) flanking the target site are created.

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Fig. Integrated IS in the chromosomal DNA

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Transposons (Tn) :

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Similar to IS elements but are more complex structurallyand carry additional genes

2 types of transposons:

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Composite transposons

Noncomposite transposons

Noncomposite transposons (Tn) :

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Carry genes (e.g., a gene for antibiotic resistance) but do notterminate with IS elements.

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 inrecombination).

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Composite transposons (Tn) :

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Carry genes (e.g., a gene for antibiotic resistance) flanked on bothsides by IS elements.

Tn10 is 9.3 kb and includes 6.5 kb of central DNA (includes a genefor tetracycline resistance) and 1.4 kb inverted IS elements.

IS elements supply transposase and ITR recognition signals.

Fig. Structures of some composite transposons

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Models of transposon transposition :

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Similar to that of IS elements; duplication at target sites occurs.

Cointegration = movement of a transposon from one genome (e.g.,plasmid) to another (e.g., chromosome) integrates transposon toboth genomes (duplication).

May be replicative (duplication) or non-replicative (transposon lostfrom original site).

Result in same types of mutations as IS elements: insertions,deletions, changes in gene expression, or duplication.

Crossing-over occurs when donor DNA with transposable elementfuses with recipient DNA.

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Advantage of Transposon mutagenesis:Easily selectable markers

Strain constructionPhysical and genetic mapping

Transposon Tn5 and Tn10 is often usedas models system

Transposon used for mutagenesis shouldhave the following properties:

High frequency of transpositionshould not very selective in its target seq

Should carry selectable marker (Antibiotic Res).Broad host range for transposition

Figure Map of pUTmini-Tn5Km1 (A), and transposon Mini-Tn5Km1 (B)

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Km1tnp *

X baIPstISphISfiI

SalI

PstIBglII

PstI

pUTmini-Tn5Km1

7.055 Kbo ri R6 K

ApR

PstIm o b RP4

SfiI EcoRI KpnI

(A)

IS IS

Not I

EcoN ISsp I

HindIIIXba I PstI Sph I Sfi I

H indIIIXh oI

S fiI EcoR I Kpn IClaI

Kan amycin R esistan ce G ene (Km 1)

1835 bp

O end(B) I end

IS (I)

R D

E. coli S17-1 λ pir(pUTmini-Tn5Km1)

M

Km 50µ g/ml

Km1tnp*

PstIX baISphISfiI

EcoR I K pnI SfiI

SalIPstI

PstI

B glII

pUTmini-Tn5Km17.055 Kb

ori R6K

Ap

PstI

pi r

Tra

Philus

MSGM agar plate

MSGMagar +

Magnetospirillum magneticum AMB-1

mini-Tn5Km1

EcoNISsp I Hind II Eco RVEcoRV HindI II

XhoI

Figure Molecular mechanism of transposon mutagenesis

primer2

DNA amplifiedDNA amplified

IS (O)

EcoRV digest

DNA genomeof mutant

Ligation(Circularization)

IS (O)IS (I)

primer1

mini-Tn 5Km1

IPCRAmplification

primer1EcoRV

primer2

Fig. The strategy for inverse PCR

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EcoRV

Primer 1 :

5'-ACACTGATGAATGTTCCGTTG-3'

Primer 2 :

5'-ACCTGCAGGCATGCAAGCTTC-3'

Number of non-magnetic mutantPosition of Mini-Tn5 (bp)

NMA35; 1014919; 911

NMA25; 1034763; 928

NMA9; 1142283; 1035

NMA59; 1376420; 1239

NMA29; 1443282; 1300

3265; 3645630; NMA42

3208; 3592045; NMA40

3149; 35294558; NMA8

3097; 3477510; NMA58

2998; 3377665; NMA20

NMA61; 1812678; 16612838; 3212025; NMA21

2575; 2880230; NMA322488; 2796870; NMA17

-; 2772204; NMA62462; 2757024; NMA51

NMA1; 1818420; 1665

NMA47; 1885655; 1725NMA41; 2185779; -

NMA10; 2211399; 2018NMA60; 2220262; 2026

NMA49; 2294609; 2085

3696; 4066333; NMA45

NMA5; 803094; 713

3712; 4095710; NMA4

NMA19; 823416; 731

4442; 4900925; NMA44

4357; 4810423; NMA564272; 4709356; NMA38

M. magneticum AMB-1

4,967,148 bp

ORF indexNMA14; 545355; 474

NMA48; 557992; 492NMA11; 558163; 492

NMA36; 635112; 557

-; 4491265; NMA34

-; 4491256; NMA334027; 4456954; NMA15

3992; 4422128; NMA50

3973; 4399224; NMA53

4967148/0

2301; 2544096; NMA13 NMA26; 2534508; 2293

Fig. Position of Mini-Tn5 insertion sites on thegenome map of M. magneticum AMB-1

Inverse PCR ofTn10 interruptedgenes

Sequence align-ment againstwhole genomesequence

Figure. Position of Tn10 insertion on the genomemap of Escherichia coli

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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.

On infection, Mu integrates by non-replicative transpostion andreplicates when E. coli replicates.

During the lytic cycle, Mu remains integrated in E. coli chromosome,and shifts to replicative transposition.

McClintock’s discovery of transposons in corn :

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Purple spots in white corn kernels are results of transposons.

c/c = white kernels and C/- = purple kernels

Kernel color alleles/traits are “unstable”.

If reversion of c to C occurs in a cell, cell will produce purplepigment and a spot.

Earlier in development reversion occurs, the larger the spot.

McClintock concluded “c” allele results from a non-autonomoustransposon called “Ds” inserted into the “C” gene (Ds =dissassociation).

Autonomous transposon “Ac” controls “Ds” transposon (Ac =activator).

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General properties of plant transposons :

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Possess ITR sequences and generate short repeats at target sites.

May activate or repress target genes, cause chromosomemutations, disrupt genes…

Two types:

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Autonomous elements transpose themselves; possesstransposition gene.

Nonautonomous elements do not transpose themselves; lacktransposition gene and reply on presence of another Tn

• McClintock demonstrated purple spots in otherwise white corn (Zea

mays) kernels are results of transposons.

Fig. 20.11, Transposon effects on corn kernel color.

McClintock’s discovery of transposons in corn (cont.) :

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Ac element is autonomous/Ds element in nonautonomous.

Ac is 4,563 bp with 11 bp ITRs and 1 transcription unit encoding an807 amino acid transposase.

Ac activates Ds; Ds varies in length and sequence, but possessessame ITRs as Ac.

Many Ds elements are deleted or rearranged version of Ac; Dselement derived from Ac.

Ac/Ds are developmentally regulated; Ac/Ds transpose only duringchromosome replication and do not leave copies behind.

Structure of Ac autonomous and Ds non-autonomoustransposable elements in corn.

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Ac transposition mechanism during chromosome replication.

Ty elements in yeast :

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Similar to bacterial transposons; terminal repeated sequences,integrate at non-homologous sites, target site duplication…

Ty elements share properties with retroviruses, retrotransposons :

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Synthesize RNA copy and make DNA using reverse transcriptase.

cDNA integrates at a new chromosomal site.

Fig. 20.14

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