transposons complete ppt

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Transposable elements

The Nobel Prize in Physiology or Medicine 1983 was awarded to Barbara McClintock "for her discovery of mobile genetic elements".

Barbara McClintock

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The Dynamic Genome

Transposons

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Transposons and Insertional Mutations Transposons: Mobile Genetic Elements

Barbara McClintock

chromosome

Transposon

Gene基因Transposon

Transposon

Mutant Gene Tagged

InsertionalMutagenesis

Transgenesis

Transposon

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Advantages of Insertional Mutations

can produce easily tractable mutations

can produce large number of mutants at low cost and high speed

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What are Transposons?Transposable element (transposon): a sequence of DNA that is competent to movefrom place to place within a genome

Some definitions and figures from Lisch 2009: Annu. Rev. Plant Biol. 2009.60:43-66.

Transposition of DNA on chromosome 9 of maize explains mottled kernels

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What are Transposons?

Transposable element (transposon): a sequence of DNA that is competent to movefrom place to place within a genome

Learn more at: weedtowonder.org/jumpingGenes.html

(1) At the beginning of kernel development, the Ds transposon is inserted into the colored (C) gene, resulting in colorless tissue. (2) Ds transposition early in kernel development restores the C gene, giving rise to a large colored sector. (3) Transposition later in kernel development results in smaller sectors.

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Transposable element (transposon): a sequence of DNA that is competent to movefrom place to place within a genome

“Cut & Paste”

“Copy & Paste”

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Autonomous element

Nonautonomous elements

Gene(s)

• Plant genomes contain multiple transposon families.

• Each contains autonomous and non-autonomous elements.• Class I transposons do not move, but are being copied.

• Class II transposons move, but can undergo copying, too (if transposing during DNA replication)


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Transposons make up the major content of eukaryotic genomes• ~50% of the genomes of human, chimp, mouse, ape

• ~75% of the maize genome

• ~85% of the barley genome

• ~98% of the iris genome

Iris brevicaulis Iris fulva

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Sorghum 700 Mb

Barley 5,000 Mb

Maize 2,500 Mb

Oats ~20,000 MbWheat 20,000 Mb

Rice 450 Mb

Variation in cereal genomes - transposons & genome duplications


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Transposons in Action

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• Most TEs are broken (cannot tranpose; “fossils”).

• Active TEs evolved to insert into “safe-havens.”

• Host regulates TE movement.

• TEs can provide advantages.

How do organisms live with TEs?

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mPing:

MITE (Multi-insertional TE)

Deletion-derivative of Ping

Requires Ping transposase to jump

MITEs are being amplified to high copy numbers

Ping/mPing

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OVER 1000 mPing copies

Japonica strains

mP

ing

cop

y n

um

ber

Naito et al PNAS (2006))

mPing

Over 1000 copies of mPing in 4 related strains….

Takatoshi Tanisaka lab (Kyoto University)

mPing copy number in O.japonica

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• predominantly in genic regions in euchromatin

• even inserts in heterochromatin are in genes

• where does mPing insert in and around genes?

Genomic distribution of mPing insertions

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0

2

4

6

8

10

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5'UTR exon intron 3'UTR

(%)

shared(n=926)

unshared(n=736)

expect.

mPing insertions rare in coding-exons

UTR Exon UTR

Genic distribution of mPing insertions

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Os02g0135500 (-41)

0

0.5

1

1.5

2

2.5

control cold salt dry

NBEG4 (mPing+)A123 (mPing+)A157

mPing found to confer cold and salt inducibility

TEs can alter gene expression

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Nipponbare EG4

EG4 is salt tolerant

TEs can alter gene expressionCan this have phenotypic consequences?

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Naito et al, Nature, 2009

• Massive amplification largely benign• Subtle impact on the expression of many genes• Produces stress-inducible networks (cold, salt, others?)• Generates dominant alleles

Rapid mPing amplification (burst)

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• TEs usually inactive.

• “Stress” conditions may activate TEs.

• Active TEs increase mutation frequency.

• Most mutations caused by TEs neutral or harmful.

• A rare TE-induced mutation (or rearrangement) may be adaptive.

Transposable elements can shake up otherwise conservative genomes and generate new genetic diversity.

TEs as tools of evolutionary change

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• (relatively) simple

• incredibly abundant

• evolve rapidly

• promote rapid genome evolution

• largely ignored (discovery)

TEs for student research projects

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Transposons Fall into two general classes with respect to

how they move. One class encodes proteins that move the

DNA element directly to a new position or replicate the DNA.– Found in both prokaryotes and eukaryotes

The other class are related to retroviruses in that they encode a reverse transcriptase for making DNA copies of their RNA transcripts, which then integrate at new sites in the genome. – Found only in eukaryotes.

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Transposable elements are important because they can insert into sites where there is no sequence homology (nonhomologous recombination)

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Prokaryotes What are two types of transposons in

prokaryotes and how do they differ? (IS and Tn)– What enzyme is required for the

transposition of an IS element?– How is a composite transposon different

from a noncomposite transposon?– How does the replicative transposition

mechanism differ from the conservative mechanism of transposition?

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EUKARYOTIC TRANSPOSITION What is cytogenetics, and how was it

used to find “jumping genes” in eukaryotes?

In what ways are eukaryotic transposable elements similar to those found in prokaryotes?

What can determine the stability of a newly-inserted transposable element in plants?

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What genes do Ty elements in yeast carry, and what are their purposes?

In what ways is the yeast Ty element similar to a retrovirus?

Why are Ty elements classified as retroposons?

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Transposable Elements (Transposons)

DNA elements capable of moving ("transposing") about the genome

Discovered by Barbara McClintock, largely from cytogenetic studies in maize, but since found in most organisms

She was studying "variegation" or sectoring in leaves and seeds

She liked to call them "controlling elements“ because they affected gene expression in myriad ways

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1. Nobelprize.org

(1983 Nobel Prize in Physiology and Medicine)

2. profiles.nlm.nih.gov/LL/

Barbara McClintock 1902-1992Corn (maize) varieties

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cob of Hopi Blue corncob of wild teosinte

Corn evolution in 7000 yrs of domestication

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Maize (domesticated corn) kernel structure

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Mutant Kernel Phenotypes1. Pigmentation mutants

– affect anthocyanin pathway– elements jump in/out of transcription

factor genes (C or R)– sectoring phenotype - somatic mutations– whole kernel effected - germ line

mutation

2. Starch synthesis mutants - stain starch with iodine, see sectoring in endosperm

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Start with lines that produce kernels defective in starch synthesis (endosperm phenotypes) or anthocyanin synthesis (aleurone and pericarp phenotypes) because of an inserted element, and the element excises during development.

Some maize phenotypes caused by transposable elements excising in somatic tissues.

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Somatic Excision of Ds from C

Fig. 23.9

SectoringWild type Mutant

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Other Characteristics of McClintock's Elements

Unstable mutations that revert frequently but often partially, giving new phenotypes.

Some elements (e.g., Ds) correlated with chromosome breaks.

Elements often move during meiosis and mitosis.

Element movement accelerated by genome damage.

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Molecular Analysis of Transposons Transposons isolated by first cloning a gene that

they invaded. A number have been cloned this way, vAia "Transposon trapping“.

Some common molecular features:– Exist as multiple copies in the genome– Insertion site of element does not have extensive

homology to the transposon– Termini are an inverted repeat– Encode “transposases” that promote movement – A short, direct repeat of genomic DNA often

flanks the transposon : “Footprint”

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Ac and Ds

Ds is derived from Ac by internal deletions Ds is not autonomous, requires Ac to move Element termini are an imperfect IR Ac encodes a protein that promotes

movement - Transposase Transposase excises element at IR, and also

cuts the target

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Structure of Ac and Ds deletion derivatives

Fig. 23.10Ds is not autonomous, requires Ac to move!

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How duplications in the target site probably occur.

Duplication remains when element excises, thus the Footprint.

Fig. 23.2

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Mu/MuDR (Mutator)

Discovered in maize; differs significantly from Ac and En/Spm families

Autonomous and non-autonomous versions; many copies per cell

Contain a long TIR (~200 bp) Transpose via a gain/loss (somatic

cells) or a replicative (germline cells) mechanism.

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Structure of MuDR (autonomous Mu) and its promoters.

• MuDrA and B expressed at high levels in dividing cells and pollen, because of transcriptional enhancers.

• MURA is transposase & has NLS.

• MURB needed for insertion in somatic cells.

47Fig. 7.34 in Buchanan et al.

Retro-Transposons

Can reach high numbers in the genome because of replicative movement.

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Control of Transposons Autoregulation: Some transposases

are transcriptional repressors of their own promoter(s)

e.g., TpnA of the Spm element

Transcriptional silencing: mechanism not well understood but correlates with methylation of the promoter (also methylation of the IRs)

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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)

or just "selfish DNA"? No known examples of an element playing a

normal role in development.

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AC and DS in maize– AC encodes transposase,

required to excise DS

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Transposon tagging

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Transposon tagging utilizes colorimetric expression assays

GUS reporter gene (B-glucuronidase), E. coli

GFP (green fluorescent protein), jellyfish

Chapter 20 slide 53

General Features of Transposable Elements1. Transposable elements are divided into two classes on the basis of their

mechanism for movement:

a. Some encode proteins that move the DNA directly to a new position or replicate the DNA to produce a new element that integrates elsewhere. This type is found in both prokaryotes and eukaryotes.

b. Others are related to retroviruses, and encode reverse transcriptase for making DNA copies of their RNA transcripts, which then integrate at new sites. This type is found only in eukaryotes.

2. Transposition is nonhomologous recombination, with insertion into DNA that has no sequence homology with the transposon.

a. In prokaryotes, transposition can be into the cell’s chromosome, a plasmid or a phage chromosome.

b. In eukaryotes, insertion can be into the same or a different chromosome.

3. Transposable elements can cause genetic changes, and have been involved in the evolution of both prokaryotic and eukaryotic genomes. Transposons may:

a. Insert into genes.

b. Increase or decrease gene expression by insertion into regulatory sequences.

c. Produce chromosomal mutations through the mechanics of transposition.

Chapter 20 slide 54

Transposable Elements in Prokaryotes

1.Prokaryotic examples include:a. Insertion sequence (IS) elements.b.Transposons (Tn).c. Bacteriophage Mu (replicated by

transposition)

Chapter 20 slide 55

Insertion SequencesAnimation: Insertion Sequences in Prokaryotes

1. IS elements are the simplest transposable elements found in prokaryotes, encoding only genes for mobilization and insertion of its DNA. IS elements are commonly found in bacterial chromosomes and plasmids.

2. IS elements were first identified in E. coli’s galactose operon, wheresome mutations’ were shown to result from insertion of a DNA sequence now called IS1 (Figure 20.1)

3. Prokaryotic IS elements range in size from 768 bp to over 5 kb. Known E. coli IS elements include:

a. IS1 is 768 bp long, and present in 4–19 copies on the E. coli chromosome.

b. IS2 has 0–12 copies on the chromosome, and 1 copy on the F plasmid.

c. IS10 is found in R plasmids.

4. The ends of all sequenced IS elements show inverted terminal repeats (IRs) of 9–41 bp (e.g., IS1 has 23 bp of nearly identical sequence).

Chapter 20 slide 56Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Fig. 20.1 The insertion sequence (IS) transposable element, IS1

Chapter 20 slide 57

5. Integration of IS elements may:

a. Disrupt coding sequences or regulatory regions.

b. Alter expression of nearby genes by the action of IS element promoters.

c. Cause deletions and inversions in adjacent DNA.

d. Serve as a site for crossing-over between duplicated IS elements.

6. When an IS element transposes:

a. The original copy stays in place, and a new copy inserts randomly into the chromosome.

b. The IS element uses the host cell replication enzymes for precise replication.

c. Transposition requires transposase, an enzyme encoded by the IS element.

d. Transposase recognizes the IR sequences to initiate transposition.

e. IS elements insert into the chromosome without sequence homology (illegitimate recombination) at target sites (Figure 20.2).

i. A staggered cut is made in the target site, and the IS element inserted.

ii. DNA polymerase and ligase fill the gaps, producing small direct repeats of the target site flanking the IS element (target site duplications).

f. Mutational analysis shows that IR sequences are the key


Fig. 20.2 Schematic of the integration of an IS element into chromosomal DNA

Chapter 20 slide 59

Transposons1. Transposons are similar to IS elements, but carry additional genes, and have a

more complex structure. There are two types of prokaryotic transposons:

a. Composite transposons carry genes (e.g., antibiotic resistance) flanked on both sides by IS elements (IS modules).

i. The IS elements are of the same type, and called ISL (left) and ISR (right).

ii. ISL and ISR may be in direct or inverted orientation to each other.

iii. Tn10 is an example of a composite transposon (Figure 20.3). It is 9.3 kb, and contains:

(1) 6.5 kb of central DNA with genes that include tetracycline resistance (a selectable marker).

(2) 1.4 kb IS elements (IS10L and IS10R) at each end, in an inverted orientation.

iv. Transposition of composite transposons results from the IS elements, which supply transposase and its recognition signals, the IRs.

(1) Tn10’s transposition is rare, because transpose is produced at a rate of ,1 molecule/generation.

(2) Transposons, like IS elements, produce target site duplications (e.g., a 9-bp duplication for Tn10). (Table 20.1)


Fig. 20.3 Structure of the composite transposon Tn10

Chapter 20 slide 61

b.Noncomposite transposons also carry genes (e.g., drug resistance) but do not terminate with IS elements.

i. Transposition proteins are encoded in the central region.

ii. The ends are repeated sequences (but not IS elements).

iii. Noncomposite transposons cause target site duplications (like composite transposons).

iv. An example is Tn3.(1) Tn3’s length is about 5 kb, with 38-bp inverted terminal

repeats.

(2) It has three genes in its central region: (a) bla encodes β-lactamase, which breaks down ampiciliin.

(b) tnpA encodes transposase, needed for insertion into a new site.

(c) tnpB encodes resolvase, involved in recombinational events needed for transposition (not found in all transposons).

(3) Tn3 produces a 5-bp duplication upon insertion (Figure 20.5).


Fig. 20.4 Structure of the noncomposite transposon Tn3


Fig. 20.5 DNA sequence of a target site of Tn3

Chapter 20 slide 64

2. Models have been generated for transposition:

a. Cointegration is an example of the replicative transposition that occurs with Tn3 and its relatives (Figure 20.6).

i. Donor DNA containing the Tn fuses with recipient DNA.

ii. The Tn is duplicated, with one copy at each donor-recipient DNA junction, producing a cointegrate.

iii. The cointegrate is resolved into two products, each with one copy of the Tn.

b. Conservative (nonreplicative) transposition is used by Tn10, for example. The Tn is lost from its original position when it transposes.

3. Transposons cause the same sorts of mutations caused by IS elements:

a. Insertion into a gene disrupts it.

b. Gene expression is changed by adjacent Tn promoters.

c. Deletions and insertions occur.

d. Crossing-over results from duplicated Tn sequences in the genome.


Fig. 20.6 Cointegration model for transposition of a transposable element by

replicative transposition

Chapter 20 slide 66

IS Elements and Transposons in Plasmids 1. Bacterial plasmids are extrachromosomal DNA capable of self-replication.

Some are episomes, able to integrate into the bacterial chromosome. The E. coli F plasmid is an example (Figure 20.7):

a. Important genetic elements of the F plasmid are: i. tra genes for conjugal transfer of DNA from donor to recipient. ii. Genes for plasmid replication. iii. 4 IS elements: 2 copies of IS3, 1 of IS2, and 1 of γδ (gammadelta). All have

homology with IS elements itt the E. coli chromosome. b. The F factor integrates by homologous recombination between IS elements,

mediated by the tra genes. 2. R plasmids have medical significance, because they carry genes for resistance to

antibiotics, and transfer them between bacteria (Figure 20.7). a. Genetic features of R plasmids include:

i. The resistance transfer factor region (RTF), needed for conjugal transfer. It includes a DNA region homologous to an F plasmid region, and genes for plasmid-specific DNA replication.

ii. Differing sets of genes, such as those for resistance to antibiotics or heavy metals. The resistance genes are transposons, flanked by IS module-like sequences, and can replicate and insert into the bacterial chromosome.

b. R plasmids are clinically significant, because they disseminate drug resistance genes between bacteria.


Fig. 20.7 Organizational maps of bacterial plasmids with transposable elements

Chapter 20 slide 68

Bacteriophage Mu 1. Temperate bacteriophage Mu (mutator) can cause mutations when it transposes.

Its structure includes:

a. A 37 kb linear DNA in the phage particle that has central phage DNA and unequal lengths of host DNA at the ends (Figure 20.8).

b. The DNA’s G segment can invert, and is found in both orientations in viral DNA.

2. Following infection, Mu integrates into the host chromosome by conservative (non-replicative) transposition.

a. Integration produces prophage DNA flanked by 5 bp target site direct repeats.

b. Flanking DNA from the previous host is lost during integration.

c. The Mu prophage now replicates only when the E. coli chromosome replicates, due to a phage-encocled repressor that prevents most Mu gene expression.

3. Mu prophage stays integrated during the lytic cycle, and replication of Mu’s genome is by replicative transposition.

4. Mu causes insertions, deletions, inversions and translocations (Figure 20.9).


Fig. 20.8 Temperate bacteriophage Mu genome shown in (a) as in phage particles and

(b) as integrated into the E. coli chromosome as a prophage


Fig. 20.9 Production of deletion or inversion by homologous recombination between

two Mu genomes or two transposons

Chapter 20 slide 71

Transposable Elements in Eukaryotes1. Rhoades (1930s) working with sweet corn, observed interactions between two

genes: a. A gene for purple seed color, the Al locus. Homozygous mutants (a/a) have colorless

seeds. b. A gene on a different chromosome, Dt (dotted) that causes seeds with genotype a/a

Dt/-- to have purple dots. i. Dt appears to mutate the a allele back to the Al wild-type in regions of the seed,

producing a dotted phenotype. ii. The effect of the Dt allele is dose dependent.

(1) One dose gave an average of 7.2 dots per seed. (2) Two doses gave an average of 22.2 dots/seed. (3) Three doses gave an average of 121.9 dots/seed.

c. Rhoades interpreted Dt as a mutator gene. 2. McClintock (1940s-50s), working with corn (Zea mays) proposed the existence

of “controlling elements” that regulate other genes and are mobile in the genome.

3. The genes studied by both Rhoades and McClintock have turned out to be transposable elements, and many others have been identified in various eukaryotes.

a. Most studied are transposons of yeast, Drosophila, corn and humans. b. Their structure is very similar to that of prokaryotic transposable elements. c. Eukaryotic transposable elements have genes for transposition and integration at a

number of sites, as well as a variety of other genes. d. Random insertion results from non-homologous recombination, and means that any

chromosomal gene may be regulated by a transposon.

Chapter 20 slide 72

Transposons in PlantsAnimation: Transposable Elements in Plants

1. Plant transposons also have IR sequences, and generate short direct target site repeats.

2. The result of transposon insertion into a plant chromosome will depend on the properties of the transposon, with possible effects including:

a. Activation or repression of adjacent genes by disrupting a cellular promoter, or by action of transposon promoters.

b. Chromosome mutations such as duplications, deletions, inversions, translocations or breakage.

c. Disruption of genes to produce a null mutation (gene is nonfunctional).

3. Several families of transposons have been identified in corn, each with characteristic numbers, types and locations.

a. Each family has two forms of transposon. Either can insert into a gene and produce a mutant allele.

i. Autonomous elements, which can transpose by themselves. Alleles produced by an autonomous element are mutable alleles, creating mutations that revert when the transposon is excised from the gene.

ii. Nonautonomous elements, which lack a transposition gene and rely on the presence of another transposon to supply the missing function. Mutation by these elements is stable (except when an autonomous element from the family is also present).

Chapter 20 slide 73

4. Multiple genes control corn color, and classical genetics indicates that a mutation in any of these genes leads to a colorless kernel. McClintock studied the unstable mutation that produces spots of purple pigment on white kernels (Figure 20.10).

a. She concluded that spots do not result from a conventional mutation, but from a controlling element (now Tn).

b. A corn plant with genotype c/c will have white kernels, while C/-- will result in purple ones.

i. If a reversion of c to C occurs in a cell, that cell will produce purple pigment, and hence a spot.

ii. The earlier in development the reversion occurs, the larger the spot.

Chapter 20 slide 74

iii. McClintock concluded that the c allele resulted from insertion of a “mobile controlling element” into the C allele.

(1) The element is Ds (dissociation), now known to be a nonautonomous transposon.

(2) Its transposition is controlled by Ac (activator), an autonomous transposon (Figure 20.11).

c. McClintock’s evidence of transposable elements did not fit the prevailing model of a static genome. More recent studies have confirmed and characterized the elements involved.

i. The Ac-Ds system involves an autonomous element (Ac) whose insertions are unstable, and a nonautonomous element (Ds) whose insertions are stable if only Ds is present.

ii. McClintock (1950s) showed that some Ds elements derive from Ac elements.


Fig. 20.11 Kernel color in corn and transposon effects

Chapter 20 slide 76

iii. Ac is 4,563 bp, with 1 1-bp imperfect terminal IRs and 1 transcription unit producing a 3.5 kb mRNA encoding an 807 amino acid transposase. Insertion generates an 8-bp target site duplication (Figure 20.12).

iv. Ac activates Ds to transpose or break the chromosome where it is inserted.

v. Ds elements vary in length and sequence, but all have the same terminal IRs as Ac, and many are deleted or rearranged versions of Ac.

vi. Unique to corn transposons, timing and frequency of transposition and gene rearrangements are developmentally regulated.

vii. Ac transposes only during chromosome replication, and does not leave a copy behind. There are two possible results of Ac transposition, depending on whether the target DNA has replicated or not (Figure 20.13). -

(1) If Ac transposes during replication into a replicated target site, its chromatid’s donor site will be empty since that copy of Ac has inserted elsewhere. In the homologous donor site on the other chromatid, a copy will remain. There is no net increase in copies of Ac.

(2) Transposition to an unreplicated chromosome site also leaves one donor site empty (and the other with a copy of Ac). The DNA into which Ac inserts will then be replicated, resulting in a net gain of one copy of Ac.

viii. Replication of Ds is the same, except that the transposition protein is supplied by an integrated Ac element.


Fig. 20.12 The structure of the Ac autonomous transposable element of corn and of

several Ds nonautonomous elements derived from Ac


Fig. 20.13 The Ac transposition mechanism

Chapter 20 slide 79

5. In Mendel’s wild-type (SS) peas the starch grains are large and simple, while in wrinlded peas (ss) they are small and fissured.

a. SS seeds contain more starch and less sucrose than ss seeds.

b. The sucrose difference makes ss seeds larger, with higher water content, so that when dried they are wrinided.

c. One type of starch-branching enzyme (SBEI) is missing in ss plants, reducing their starch content.

d. The SBEI gene corresponding to the s allele has a 0.8 kb transposon similar to the Ax/Ds family inserted into the wild-type S allele.

Chapter 20 slide 80

Ty Elements in Yeast 1. Ty elements share characteristics with bacterial transposons:

a. Terminal repeated sequences. b. Integration at non-homologous sites. c. Generation of a target site duplication (5 bp).

2. Ty element is diagrammed in Figure 20.14: a. It is 5.9 kb including 2 terminal direct repeats of 334 bp, the long terminal repeats

(LTR) or deltas (δ). b. Each delta contains a promoter and transposase recognition sequences. c. Ty elements encode one 5.7 kb mRNA beginning at the delta 5’ promoter (Figure

20.14). d. There are two ORFs in the mRNA, designated TyA and TyB, encoding two different

proteins. e. Ty copy number varies between yeast strains, with an average of about 35.

3. Ty elements also share similarities with retroviruses, ssRNA viruses that replicate via dsDNA intermediates.

a. Ty elements transpose by making an RNA copy of the integrated DNA sequence, them making DNA using reverse transcriptase. This DNA can integrate at a new chromosomal site. Evidence for this includes:

i. An experimentally introduced intron in the Ty element (which normally lacks introns) was monitored through transposition. The intron was removed, indicating an RNA intermediate.

ii. Ty elements encode a reverse transcriptase. iii. Virus-like particles containing Ty RNA and reverse transcriptase activity occur.

b. Ty elements are referred to as retrotransposons.


Fig. 20.14 The Ty transposable element of yeast

Chapter 20 slide 82

Drosophila transposons 1. It is estimated that 15% of the Drosophila genome is mobile! These

transposons fall into different classes:

a. The copia retrotransposons include several families, each highly conserved and present in 5-100 widely scattered copies per genome (Figure 20.15).

i. All copia elements in Drosophila can transpose, and there are differences in number and distribution between fly strains.

ii. Structurally, copia elements are similar to yeast Ty elements:

(1) Direct LTRs of 276 bp flank a 5 kb DNA segment.

(2) The end of each LTR has 17 bp inverted repeats.

(3) An RNA intermediate and reverse transcriptase are used for transposition.

(4) Virus-like particles (VLPs) occur with copia.

(5) Integration results in target site duplication (3-6 bp).


Fig. 20.15 Structure of the transposable element copia, a retrotransposon found in

Drosophila melanogaster

Chapter 20 slide 84

b. P elements cause hybrid dysgenesis, a series of defects (mutations, chromosomal aberrations and sterility) that result from crossing certain Drosophila strains (Figure 20.16).

i. A mutant lab strain female (M) crossed with a wild-type male (P) will result in hybrid dysgenesis.

ii. A mutant lab strain male (M) crossed with a wild-type (P) female (reciprocal cross) will have normal offspring.

iii. Thus, hybrid dysgenesis results when chromosomes of the P male parent enter cytoplasm of an M type oocyte, but cytoplasm from P oocytes does not induce hybrid dysgenesis.


Fig. 20.16 Hybrid dysgenesis, exemplified by the production of sterile flies

Chapter 20 slide 86

iv. The model is based on the observation that the M strain has no P elements, while the haploid genome of the P male has about 40 copies.

(1) P elements vary from full-length autonomous elements through shorter versions resulting from a variety of internal deletions.

(2) P element transposition is activated only in the germ line. (3) The F1 of an M female crossed with a P male have P

elements inserted at new sites, flanked by target site repeats. (4) P elements are thought to encode a repressor protein that

prevents transposase gene expression, preventing transposition.

(5) Cytoplasm in an M oocyte lacks the repressor, and so when fertilized with P-bearing chromosomes, transposition occurs into the maternal chromosomes, leading to hybrid dysgenesis.

v. P elements are used experimentally to transfer genes into the germ line of Drosophila embryos. For example (Figure 20.18):

(1) The wild-type rosy (ry) gene was inserted into a P element, cloned in a plasmid and microinjected into a mutant ry/ry strain.

(2) Insertion of the recombinant P element into the recipient chromosome introduced the ry allele, and produced wild-type flies.


Fig. 20.17 Structure of the autonomous P transposable element found in Drosophila

melanogaster


Fig. 20.18 Illustration of the use of P elements to introduce genes into the Drosophila

genome

Chapter 20 slide 89

Human Retrotansposons 1. Retrotransposons also appear to be present in mammals. For example, a

very abundant human SINE repeat (short interspersed sequence) is the Mu family, named for the AluI restriction site in its sequence. a. Mu sequences are about 300 bp, repeated 300,000-500,000 times in the human

genome (up to 3% of total human DNA). b. Sequences are divergent, related but not identical. c. Each Mu sequence is flanked by 7-20 bp direct repeats. d. At least a few Mu sequences can be transcribed, and the model is that

transcriptionally active Mu sequences are retrotransposons that move via an RNA intermediate.

e. A human case of a genetic disease, neurofibromatosis, provides some evidence. i. Neurofibromas (tumorlike growths on the body) result from an autosomal

dominant mutation. ii. In a patient’s DNA, an unusual Mu sequence was detected in one of the

introns of the neurofibromatosis gene. iii. The resulting longer transcript is incorrectly proessed, removing an exon

from the mRNA and producing a nonfunctional protein. iv. Neither parent had this Mu sequence in the neurofibromatosis gene. v. Divergent Mu sequences made it possible to track this particular version to

an insertion event in the germ line of the patient’s father. f. It is not clear how the functions needed for Mu retrotransposition are provided.

Chapter 20 slide 90

2. A mammalian LINEs family, LINEs-i (Li elements) is also thought to be retrotransposons.

a. Humans have 50,000-100,000 copies of the Li element, comprising about 5% of the genome.

b. The full-length element (6.5 kb) is not abundant, and most Li elements are deleted versions.

c. The full-length Li element contains a large ORF with homolegy to known reverse transcriptases. Experimentally, the Li ORF can substitute for the yeast Ty reverse transcriptase gene.

d. Li elements are thought to be retrotransposons, but do not have LTRs.

e. Clinically, cases of hemophilia have been shown to result from newly transposed Li insertions into the factor VIII gene. (Factor VIII is required for normal blood clotting.)

91

21.1 Introduction

Figure 21.1

92

21.2 Insertion Sequences Are Simple Transposition

Modules

An insertion sequence is a transposon that codes for the enzyme(s) needed for transposition flanked by short inverted terminal repeats.

93

The target site at which a transposon is inserted is duplicated during the insertion process.– This forms two repeats

in direct orientation at the ends of the transposon.

The length of the direct repeat is:– 5 to 9 bp– characteristic for any

particular transposonFigure 21.2

94

21.3 Composite Transposons Have IS Modules

Transposons can carry other genes in addition to those coding for transposition.

Composite transposons have a central region flanked by an IS element at each end.

95

Either one or both of the IS elements of a composite transposon may be able to undertake transposition.

A composite transposon may transpose as a unit.– An active IS element at

either end may also transpose independently.

Figure 21.3

96

21.4 Transposition Occurs by Both Replicative and

Nonreplicative Mechanisms All transposons use

a common mechanism in which:– staggered nicks are

made in target DNA– the transposon is

joined to the protruding ends

– the gaps are filledFigure 21.5

97

The order of events and exact nature of the connections between transposon and target DNA determine whether transposition is:– replicative– nonreplicative

Figure 21.7Figure 21.6

98

21.5 Transposons Cause Rearrangement of DNA

Homologous recombination between multiple copies of a transposon causes rearrangement of host DNA.

Homologous recombination between the repeats of a transposon may lead to precise or imprecise excision.

99

21.6 Common Intermediates for

Transposition Transposition starts by

forming a strand transfer complex.– The transposon is

connected to the target site through one strand at each end.

Figure 21.11

100

The Mu transposase forms the complex by:– synapsing the ends of Mu

DNA– followed by nicking– then a strand transfer

reaction

Replicative transposition follows if the complex is replicated.– Nonreplicative transposition

follows if it is repaired.Figure 21.12

101

21.7 Replicative Transposition Proceeds through a Cointegrate

Replication of a strand transfer complex generates a cointegrate:– A fusion of the donor and

target replicons.

The cointegrate has two copies of the transposon.– They lie between the

original replicons.Figure 21.13

102

Recombination between the transposon copies regenerates the original replicons, but the recipient has gained a copy of the transposon.

The recombination reaction is catalyzed by a resolvase coded by the transposon.

103

21.8 Nonreplicative Transposition Proceeds by

Breakage and Reunion Nonreplicative transposition results if:

– a crossover structure is nicked on the unbroken pair of donor strands and

– the target strands on either side of the transposon are ligated

Figure 21.15

104

Two pathways for nonreplicative transposition differ according to whether:

– the first pair of transposon strands are joined to the target before the second pair are cut (Tn5), or

– whether all four strands are cut before joining to the target (Tn10)

105

21.9 TnA Transposition Requires Transposase and

Resolvase Replicative transposition of TnA requires:

– a transposase to form the cointegrate structure– a resolvase to release the two replicons

The action of the resolvase resembles lambda Int protein.

It belongs to the general family of topoisomerase-like site-specific recombination reactions.– They pass through an intermediate in which the

protein is covalently bound to the DNA.

106

21.10 Transposition of Tn10 Has Multiple Controls

Multicopy inhibition reduces the rate of transposition of any one copy of a transposon when other copies of the same transposon are introduced into the genome.

Multiple mechanisms affect the rate of transposition.

Figure 21.21

107

21.11 Controlling Elements in Maize Cause Breakage

and Rearrangements Transposition in maize was discovered

because of the effects of chromosome breaks.– The breaks were generated by transposition

of “controlling elements.”

The break generates one chromosome that has:– a centromere– a broken end – one acentric fragment

108

The acentric fragment is lost during mitosis; – this can be detected

by the disappearance of dominant alleles in a heterozygote.

Figure 21.23

109

Fusion between the broken ends of the chromosome generates dicentric chromosomes.– These undergo further cycles

of breakage and fusion.

The fusion-breakage-bridge cycle is responsible for the occurrence of somatic variegation.

Figure 21.24

110

21.12 Controlling Elements Form Families of

Transposons

Each family of transposons in maize has both autonomous and nonautonomous controlling elements.

Figure 21.25

111

Autonomous controlling elements code for proteins that enable them to transpose.

Nonautonomous controlling elements have mutations that eliminate their capacity to catalyze transposition.– They can transpose when an autonomous

element provides the necessary proteins.

Autonomous controlling elements have changes of phase, when their properties alter as a result of changes in the state of methylation.

112

21.13 Spm Elements Influence Gene Expression

Spm elements affect gene expression at their sites of insertion, when the TnpA protein binds to its target sites at the ends of the transposon.

Spm elements are inactivated by methylation.

113

21.14 The Role of Transposable Elements in

Hybrid Dysgenesis P elements are transposons that are

carried in P strains of Drosophila melanogaster, but not in M strains.

When a P male is crossed with an M female, transposition is activated.

114

The insertion of P elements at new sites in these crosses:– inactivates many genes– makes the cross infertile

Figure 21.28

115

21.15 P Elements Are Activated in the Germline

P elements are activated in the germline of P male x M female crosses.

This is because a tissue-specific splicing event removes one intron.– This generates the

coding sequence for the transposase.

Figure 21.29

116

The P element also produces a repressor of transposition.– It is inherited

maternally in the cytoplasm.

The presence of the repressor explains why M male x P female crosses remain fertile.Figure 21.30

Pray, L. (2008) Transposons: The jumping genes. Nature Education 1(1)

DNA transposons Seen in both prokaryotes and

eukaryotes– the IS element (insertion sequence) in bacteria– DNA transposons seen in eukaryotic genomes (P

elements in fruit flies, Ac/Ds elements in plant genomes)

Mechanism of transposon action– Transposon encodes an enzyme: transposase– Transposase excises itself out and then is able to

cut in the middle of a target DNA– Effect is based on where the transposable element

inserts– Insertion identified by the chararcteristic flanking

direct and indirect repeats

RNA transposable elements

Derived from an RNA intermediate Seen only in eukaryotic genomes Originated from ancient retroviral

genome– Retrotransposon

LTR elements– Retroposons

SINE-human LINE-human

- Derived from a viral genome from the retrovirus:

LTR LTRgag RT env

~7 kbRT: reverse transcriptaseLTR: long terminal repeatgag, env: encode proteins needed for retroviral assembly and infection

Retroelements: missing some or most of the complete retroviral genome;

LTR LTRgag RT

~7 kb

- Retrotransposons:contain the LTR repeats

- make up ~50% of the

maize genome

Mechanism of retrotransposition RNA

Retrotransposon

Transcription

RNAReverse transcription

DNA

Retrotransposon

Retrotransposon copy

Human Retroposons: non-LTR - LINE: long interspersed elements

~6 kb

poly(A)gag? RT

- SINE: short interspersed element; The Alu element is a well known example

~0.3 kb

poly(A)

C-value paradox: genome size not always predictor of gene number

Taken fron http://cs.uni.edu

Transposable Elements

DNA Sequences That Change Positions in the Genome

Types of Transposable Elements

Type Transposition Examples

Transposon(Class I)

Use a DNA intermediate

Corn: Ds elementDrosophila: P element

Retrotransposons(Class II)

Use an RNA intermediate

Yeast: TyDrosophila: Copia Human: Alu Human: L1

Transposition: movement of a transposable element

Characteristics of Transposable Elements

All elements have direct repeats: short repeated sequences flanking the element, arise by transposition


Some elements have terminal inverted repeats


Carry gene for enzyme that catalyzes transposition– transposase for elements that use a

DNA intermediate– reverse transcriptase for elements

that use an RNA intermediate May contain other genes

Mechanisms of Transposition Use of a DNA Intermediate

– Replicative- new copy in new location, old copy retained at original site, element is used as template to produce the new copy

Mechanisms of Transposition Use of a DNA Intermediate

– Non-replicative: moves to another site without replication of the element

Mechanisms of Transposition

Use of an RNA Intermediate– element is

transcribed– reverse

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

Types of Retrotransposons Viral Retrotransposons

– resemble retroviruses = viruses with an RNA genome

Long terminal direct repeat at each end Carry genes for enzymes usually found in

RNA viruses

Retrovirus Characteristics

Types of Retrotransposons Non-viral Retrotransposons

– do not resemble retroviruses– two types in humans

LINES = long interspersed elements– 6-7 kb long– example: L1 has 600,000 copies, represents 15%

of human DNA SINES = short interspersed elements

– 300 bp long – example: Alu has 1 million copies, represents

10% of human DNA

Definitions and Keywords Transposons - are sequences of

DNA that can move around to different positions within the genome of a single cell, a process called transposition.

Transposase -An enzyme that binds to ends of transposon and catalyses the movement of the transposon to another part of the genome by a cut and paste mechanism or a replicative transposition mechanism.

IS elements -A short DNA sequence that acts as a simple transposable element

Definitions and Keywords

DNA polymerase-A DNA polymerase is an enzyme that catalyzes the polymerization of deoxyribonucleotides into a DNA strand.

DNA ligase is a special type of ligase that can link together two DNA strands that have double-strand break a break in both complementary strands of DNA

http://en.wikipedia.org/wiki/Polymerase

http://en.wikipedia.org/wiki/Enzyme

http://en.wikipedia.org/wiki/Catalyze

http://en.wikipedia.org/wiki/Polymerization

http://en.wikipedia.org/wiki/Deoxyribonucleotide

http://en.wikipedia.org/wiki/DNA

http://en.wikipedia.org/wiki/Ligase

http://en.wikipedia.org/wiki/DNA

Bacterial Transposons Bacteria contain two types of

transposons

1]Composite mobile genetic elements that are larger than IS elements and contain one or more protein-coding genes in addition to those required for transposition.

2]Non composite mobile genetic elements are those which lack IS elements on its ends e.g. is Tn3

Transposone

Presented by

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

Jumping genes Selfish DNAs Molecular parasites Controlling elements

TEs are present in the genome all species of three domains

Transposable Elements

What do we want to know about mobile genetics elements?

1 – The history of mobile genetic elements

2 – The classification of TEs 3 – The structure of TEs 4 – The mechanism of

transposition 5 – The effects of TEs on gene and

genome 6 – The use of TEs as molecular

tools

BACTERIAL TRANSPOSONS

TRANSPOSONS “Transposable elements” “Jumping genes” Mobile DNA

– able to move from one place to another within a cell’s genome

– sometimes a copy is made and the copy moves

– insertion requires target DNA sequences

Transposon

inverted terminal

repeat (ITR)

In the process, they may - cause mutations. - increase (or decrease) the

amount of DNA in the genome. - promote genome

rearrangements. - regulate gene expression. - induce chromosome breakage and rearrangement.

Discovery of transposons Barbara McClintock 1950’s Ac Ds

system in maize influencing kernel color unstable elementschanging map position promote chromosomal breaks.

Rediscovery of bacterial insertion sequencessource of polar mutations discrete change in physical length of DNA inverted repeat ends: form “lollipops” in EM after denaturation.

These mobile segments of DNA are sometimes

called "jumping genes"

There are two distinct types of transposons:

1) DNA transposons -transposons consisting only of DNA that

moves directly from place to place 2) Retrotransposons - first transcribe the DNA into RNA and

then - use reverse transcriptase to make a DNA

copy of the RNA to insert in a new location

Classification of Transposons into two classes

In both cases ds

DNA intermediate

is integrated into

the target site in

DNA to complete

movement

BACTERIAL TRANSPOSONS

● In bacteria, transposons can jump from chromosomal DNA to plasmid DNA and back.

● Transposons in bacteria usually

carry an additional gene for function other than transposition---often for antibiotic resistance.

● Bacterial transposons of this type

belong to the Tn family. When the transposable elements lack additional genes, they are known as insertion sequences.

BACTERIAL TRANSPOSONS - TYPES

1. Insertion sequence2.Composite transposon3.Tn3-type transposon4.Transposable phage

1.Insertion sequences

Insertion sequences – IS1 and IS186, present in the 50-kb segment of the E. coli DNA, are examples of DNA transposons.

Single E. coli genome may contain 20 of

them. Most of the sequence is taken by one or two

genes for transposase enzyme that catalyses transposition.

IS elements transpose either replicatively or conservatively.

cont….

IS elements Study of E. coli mutations resulting from insertion of 1-2 kb

long sequence in the middle of certain genes.

Inserted stretches or insertion sequences – could be visualized by EM.

IS - molecular parasites in bacterial cells.

Transposition of IS is very rare – one in 105-107 cells per generation.

Higher rates result in greater mutation rates.

Bacterial IS element

Central region encodes for one or two enzymes required for transposition. It is flanked by inverted repeats of characteristic sequence.

The 5’ and 3’ short direct repeats are generated from the target-site DNA during the insertion of mobile element.

The length of these repeats is constant for a given IS element, but their sequence depends upon the site of insertion and is not characteristic for the IS element.

Arrows indicate orientation.

Insertion sequences in E.coli

Elements Size (bp) No.of.copies/ genome

IS1 768 8

IS2 1327 5

IS3 1300 1 or more

IS4 1426 1 or more

2.Composite transposons

Bacteria contain composite mobile genetic elements that are larger than IS elements and contain one or more protein-coding genes in addition to those required for transposition:

Composite transposons - are basically the pair of IS elements flanking a segment of DNA usually containing one or more genes, often coding for AB resistance.

They use conservative method of

transposition.

Cont… 2.Composite transposon - Antibiotic resistant gene - Flank by IS element (inverted or directed repeat)

- Terminal IS can transpose by in selfEx. Tn5, Tn9, Tn10

3. Tn 3 transposon family

- 5000 bp - code for Transposase, β-

lactamase, Resolvase - Function of resolvase Decrease Transposase

production Catalyse the

recombination of transposon

Cont…

Tn3 – type transposon --- 5kb ITR - inverted terminal repeat β- lactamase – Resistance gene

ITRITR

resolvasetransposase β-lactamase

4.Transposable phage Transposable phages –

bacterial viruses which tranpose replicatively as a part of their normal infectious cycle.

Integrate into E. coli chromosome at regulatory element

Eg. Mu phage

Transposable phage

Transposable phage – 38kb ITR - inverted terminal repeats

ITRITR

Lysis genesIntegration and Replication genes

Protein coatgenes

Transposable phage - Mu phage

Mechanism of transposition

Two distinct mechanisms of transposition:

Replicative transposition – direct

interaction between the donor transposon and the target site, resulting in copying of the donor element

Conservative transposition –

involving excision of the element and reintegration at a new site.

Mechanism of transposition1. Replicative transposition

Copy of transposon sequence

Transposase enzyme cut target DNA

Transposition

Duplication of target sequence

Replicative transposition

2. Non-replicative (conservative)transposition

- Cannot copy transposon sequence

- Transposition by cut and paste model

Cut transposon sequence from donor molecule

attach to target site Ex. IS10, Tn10

Non-replicative (conservative) transposition

Evolution of Transposons Transposons are found in all major

branches of life.

It arisen once and then spread to other kingdoms by horizontal gene transfer.

Duplications and DNA rearrangements contributed greatly to the evolution of new genes.

Cont…

Mobile DNA most likely also influenced the evolution of genes that contain multiple copies of similar exons encoding similar protein domains (e.g., the fibronectin gene).

The evolution of an enormous variety of antibiotic resistance transposons and their spread among bacterial species.

example of genetic adaptation via natural selection.

Transposons causing diseases

Transposons are mutagens. They can damage the genome of their host cell in different ways:

1. A transposon or a retroposon that inserts itself

into a functional gene will most likely disable that gene.

2.After a transposon leaves a gene, the resulting gap will probably not be repaired correctly.

3.Multiple copies of the same sequence, such as Alu sequences can hinder precise chromosomal pairing during mitosis and meiosis, resulting in unequal crossovers, one of the main reasons for chromosome duplication.

Cont… Diseases caused by transposons

include -hemophilia A and B -severe combined

immunodeficiency -Porphyria -Cancer -Duchenne muscular dystrophy

Applications

The first transposon was discovered in the plant maize (Zea mays, corn species), and is named dissociator (Ds).

Likewise, the first transposon to be molecularly isolated was from a plant (Snapdragon).

Transposons have been an especially useful tool in plant molecular biology.

Researchers use transposons as a means of mutagenesis.

Cont… To identifying the mutant allele.

To study the chemical mutagenesis methods.

To study gene expression.

Transposons are also a widely used tool for mutagenesis of most experimentally tractable organisms.

QUERIES ?

Why study mobile genetic elements?

They are the major forces driving evolution

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

They have wide range of application potentials

The discovery of mobile genetic elements


Phage

Plasmid DNA

The discovery of transposable elements Barbara Mc Clintock discovered TEs in

maize (1983)

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

Observing variegated patterns of pigmentation in maize plant and kernels

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

genetic elements

Controlling elements (1956)

Barbara Mc Clintock 1902 1980 ( noble in 1984)

Plasmid , phage Cell to cell conjugation Bactriophage mediated

transduction Bill Hayes ( 1952 ) Ellin Wollman and Francois Jancob

, 1961 Alan Campbell

Classification of transposable elements

DNA transposons Retrotransposons

Autonomous and non autonomous elements

Both class are subdivided into distinct superfamilies and families

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

Autonomous : they have the ability to excise and transpose

non autonomous elements They don’t transpose They become unstable only when an autonomous

member of same family is present elsewhere in the genome

They are derived from autonomous elements

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

DNA based elements Insertion sequence (IS) The simplest (smallest)

transposons are called IS The IS elements are normal

constituents of bacterial chromosome and plasmids

Spontaneous mutation of the lac and gal operons

They are autonomous units ,each of which codes only transposase

Structure of IS

Composite transposone One class of large

transposons are called Composite transposons

They carring the druge marker is flanked on either side by arms 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


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 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

Donor cut

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

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 cut

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

Cutting of both ends

3 ‘ OH

3 ‘ OH

3 ‘ OH

3 ‘ OH

Cutting of 3 ‘ end only

The product of these reaction is 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 )

Use of an RNA Intermediate– element is transcribed– reverse transcriptase

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

– they as other elements generating short direct repeat

Types of Retrotransposons

1 – viral superfamily (autonomousretrotransposon)

– retrovirus – LTR- retrotransposon – LINES

2 – nonviral superfamily (non autonomous retransposons)

SINES

non LTR- retrotransposon

retrovirus

RNA

reverstranscriptase

Liner DNA

Integration

provirus

Transcription

RNA

LTR - retrotrasposon

pol

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’ -ends and staggered end in target DNA

3’-ends5’-ends

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

sequence of mammalian genomes .

Effect of transposabli elements on gene and genome

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

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

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

play important role in evolution .

they have the ability to rearrange genomic information in several ways

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

Modification of gene expression

insertion of a TE within or adjacent to a gene

the element blocks or alters the pattern of transcription .

insertion of Fot1 in a intron of niad (F . oxysporum )

different mutant transcripts all were shorter

They result from: - presence of termination signal - presence of an alternative

promotor

Alternation gene sequence

cut and pate mechanism often produce variation when they excise .

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

these footprint cause diversification of nucleotide sequence and new functional alleles

Example :Fot1 and Impala generally leave 4 bp – ( 108 ) or 5 – ( 63 ) foot prints

excision of the Asco - 1 transposon in A .immersus Deletions of a a few to up to 1713 nucleotide in b2

gene larger deletion led to variety of phenotypes in spore

coloration

Chromosomal structural changes

TEs can produce a series of genome rearrangment through ectopic recombination

deletion , duplication , inversion and translucation mediate by TEs ( Drosophila , Yeast , human )

karyoptypic variation in natural isolate in fungai

high level of chromosome – length polymorphism (Magnoporthe grisea , F. oxysporum)

translocation tox1 of Cochliobolus heterostrophus

appearance of new virulence alleles in M . grisea

Use as strain specific diagnostic tools

TEs are often restricted to specific strains

identify specific pathogen in plant pathology

Fot1 ( F. oxysporum f sp. albedians ) provide PCR targets

a sensitive detection thechnique to prevent the introduction of pathogenic form

- race of F. oxysporum responsible of carnation wilt

- date palm pathogen

Use of TEs as molecular tools


MGR 586 ( Magneporthe grisea ) oryza : 30 – 50 wheat and other

( 1 – 2 ) they have used to distinguish

genetically divergent population fingerprinting of isolates

pathogenic to oil palm tree. ( F. oxysporum, palm)

Tools for the analysis of population structure

Gene tagging with transposable elements

arise mutant phenotype

Disrupt target gene


jumping into coding region

Target gene can easily determined by PCR methods

Target gene can easily determined by PCR methods

Thanks for attention

Composite TransposonA composite transposon, is flanked by two separate IS elements which may or may not be exact replicas. Instead of each IS element moving separately, the entire length of DNA spanning from one IS element to the other is transposed as

one complete unit.

IR IR

Non composite Transposon

Non-composite transposons (which lack flanking insertion sequences). In each case, transposition requires specific DNA sequences located at the ends (IS1, IS3, Tn5, Tn10, and Tn3) or a multisubunit complex (e.g. Tn7).

Encode transposition proteins, have inverted repeats (but no ISs) at their ends. In addition to resistance and virulence genes they may encode catabolic enzymes


There are two mechanisms of transposition replicative and nonreplicative

During transposition, the IS-element transposase makes cuts at the positions indicated by small red arrows,

So the entire transposon is moved from the donor DNA (e.g., a plasmid).

A DNA polymerase fills in the resulting gaps from the sticky ends and DNA ligase closes the sugar-phosphate backbone. This results in target site duplication and the insertion sites of DNA transposons may be identified by short direct repeats (a staggered cut in the target DNA filled by DNA polymerase) followed by inverted repeats (which are important for the transposon excision by transposase). The duplications at the target site can result in gene duplication and this is supposed to play an important role in evolution.

Composite transposons will also often carry one or more genes conferring antibiotic resistance

http://en.wikipedia.org/wiki/DNA_polymerase

http://en.wikipedia.org/wiki/DNA_ligase

http://en.wikipedia.org/wiki/Gene_duplication

http://en.wikipedia.org/wiki/Evolution

Mechanism of transposition(contd)

The conservative mechanism, also called the “cut-and-paste” mechanism, is used by elements like Tn10 .

The element is excised cleanly by double-strand cleavages from the donor DNA

and inserted, with limited repair, between a pair of staggered nicks at the target DNA.

Replicative transposition is a mechanism of transposition in molecular biology in which the transposable element is duplicated during the reaction, so that the transposing entity is a copy of the original element. Replicative transposition is characteristic to retrotransposons and occurs from time to time in class II transposons.

Retrieved from "http://en.wikipedia.org/wiki/Replicative_transposition

http://en.wikipedia.org/wiki/Molecular_biology

http://en.wikipedia.org/wiki/Transposable_element

http://en.wikipedia.org/wiki/Retrotransposon

http://en.wikipedia.org/wiki/Replicative_transposition

http://en.wikipedia.org/wiki/Replicative_transposition

General mechanism of Transposition

Production of protein (enzyme transposase) from the site of transposase(right corner an Tn 5) should be shown.{the site in upper diagram in between IR of IS element.}Action/Motion-Production of protein (enzyme transposase) from the site of transposase (right corner an Tn 5) should be shown

http://upload.wikimedia.org/wikipedia/commons/5/57/Transposase.png

Replicative TranspositionSingle stranded cuts are made on either side of the Transposon and on the opposite sides of the target of the recipient.

getThis produces 4 free ends in each DNA moleculeTwo of the ends from the donor are ligated to 2 of the ends of target.

This links the two molecules with a single molecule of transposon.

The two remaining free 3’ ends are used as primers for DNA polymerase which uses the Transposon DNA as the template.This replicates the transposon and leaves the cointegrate.

NickingSingle strranded cuts produce staggered ends in both transposon and target

Crossover structure (strand transfer complex)Nicked ends of Transposon are joined to nicked ends of target.

Replication from free 3’ end generate cointegrate

Single molecule has two types of transposon.

Cointegrate drawn as continuous path shows that transposons are at junctions between replicons.

NON REPLICATIVE TRANSPOSON

First, the transposase makes a double-stranded cut in the donor DNA at the ends of the transposon and makes a staggered cut in the recipient DNA.

Each end of the donor DNA is then joined to an overhanging end of the recipient DNA.

DNA polymerase fills in the short,

overhanging sequences,

resulting in a short, direct repeat

on each side of the transposon

insertion in the recipient DNA.

INSTRUCTIONS SLIDE

Questionnaire to test the userQ1]Define tranposition?Transposons are sequences of DNA that can move around

to different positions within the genome of a single cell, a process called transposition.

Q2]Give examples of non composite transposons.IS1, IS3, Tn5, Tn10, and Tn3) or a multisubunit complex (e.g.

Tn7)Q3]Describe the general structure of bacterial transposons.Ans

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This transposon consists of a chloramphenicol-resistance gene (dark blue) flanked by two copies of IS1 (orange), one of the smallest IS elements. Other copies of IS1, without the drug-resistance gene, are located elsewhere in the E. coli chromosome. The internal inverted repeats of IS1 abutting the resistance gene are so mutated that transposase does not recognize them. During transposition, the IS-element transposase makes cuts at the positions indicated by small red arrows, so the entire transposon is moved from the donor DNA (e.g., a plasmid). The target-site sequence at the point of insertion becomes duplicated on either side of the transposon during transposition, which occurs via the replicative mechanism. Note that the 5-bp target-site direct repeat (light blue) is not to scale

Q4]Explain the mobile genetic elements found in bacteria.ANS:-

Three of the many types of mobile genetic elements found in bacteria. Each of these DNA elements contains a gene that encodes a transposase, an enzyme that conducts at least some of the DNA breakage and joining reactions needed for the element to move. Each mobile element also carries short DNA sequences (indicated in red) that are recognized only by the transposase encoded by that element and are necessary for movement of the element. In addition, two of the three mobile elements shown carry genes that encode enzymes that inactivate the antibiotics ampicillin (ampR) and tetracycline (tetR). The transposable element Tn10, shown in the bottom diagram, is thought to have evolved from the chance landing of two short mobile elements on either side of a tetracyclin-resistance gene; the wide use of tetracycline as an antibiotic has aided the spread of this gene through bacterial populations. The three mobile elements shown are all examples of DNA-only transposons

Q5]Illustrate the mechanism of transposition in transposons.

ANS:-

Links for further reading1

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Molecular Cell BiolOGYBaltimore-molecUlar biology of the gene watson-Genes Lewin -VOET AND VOET-LEHNINGER-COOPER

Thank you

Applying Your Knowledge

Which type of transposable element• Uses a DNA intermediate for transposition?• Contains long terminal repeats on its ends? • Generates direct repeats as a result of

transposition?• Carries a gene for reverse transcriptase?• Can insert a copy in a new location while leaving

the old copy at the original site?

1. Retrotransposon2. Transposon3. Both retrotransposons and transposons4. Neither retrotransposons nor transposons

Effects of Transposition

Transposable elements can:

Cause mutations in adjacent genes

Cause chromosomal rearrangements

Relocate genes

Possible Advantages of Transposable Elements

Transposable elements may: Create genetic diversity Act as promoters Allow recombination between

plasmid and genomic DNA when multiple copies of the element are present

Carry antibiotic resistance genes, conferring an advantage on bacterial cells

Increase the number of copies of an exon or gene

Examples of Transposable Elements

Bacterial Insertion Sequences and

more Complex Transposons Ac-Ds Elements in Corn P elements in Fruit Flies

Transposable Elements in Bacteria

Insertion Sequences contain only the elements needed for transposition

Composite Transposons contain DNA that has insertion sequences on both sides

Antibiotic resistance genes are often included

Ac and Ds Elements in Corn

Ac stands for activator element Ds stands for dissociative element Barbara McClintock showed that

--transposition of the Ds element altered kernel coloration

--movement of the Ds element required the activity of Ac element

Animation available at http://www.dnalc.org

Transposition of Ds Element Disrupts Gene Controlling Kernel

Color

Excision of Ds Element Leads to Variegated Kernels

Relatedness of Ac and Ds Elements

For transposition, Ds elements require the transposase produced by the Ac element.

Autonomous and Non-autonomous Elements

Type Properties Example

Autonomous •Can transpose without the presence of another element

Non-autonomous

•Requires the presence of another functional element to move•Autonomous element provides transposase or reverse transriptase

Ac

Ds

The P Element in Drosophila Codes for a Transposase and a Repressor of

Transposition

No repressor

P element inserts in multiple

locations

Repressor produced

Transposition is repressed

Use of the P Element As a Vector in Drosophila

P element codes for transposase

P element with gene of interest can insert into chromosomeswith help of plasmid containing only transposase.

Applying Your Knowledge

Which type of transposable element• Contains only the sequences needed for

transposition in bacteria?• Represents a SINE found in humans? • Is used to insert genes into fruit fly

chromosomes?• Causes reversible alterations for kernel color

in corn?

1. Ac-Ds Elements2. Alu Element3. Insertion Sequence4. P element

transposons complete ppt

Education

active tes

tes neutral

copies of mping

a123 mping

sequence of dna

genome transposition

rapid mping amplification

mping copy number