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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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What are Transposons?
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)
What are Transposons?
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What are Transposons?
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
What are Transposons?
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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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Transposable elements
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
Chapter 20 slide 58Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
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)
Chapter 20 slide 60Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
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).
Chapter 20 slide 62Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Fig. 20.4 Structure of the noncomposite transposon Tn3
Chapter 20 slide 63Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
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.
Chapter 20 slide 65Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
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.
Chapter 20 slide 67Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
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).
Chapter 20 slide 69Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
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
Chapter 20 slide 70Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
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.
Chapter 20 slide 75Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
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.
Chapter 20 slide 77Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Fig. 20.12 The structure of the Ac autonomous transposable element of corn and of
several Ds nonautonomous elements derived from Ac
Chapter 20 slide 78Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
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.
Chapter 20 slide 81Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
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).
Chapter 20 slide 83Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
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.
Chapter 20 slide 85Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
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.
Chapter 20 slide 87Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Fig. 20.17 Structure of the autonomous P transposable element found in Drosophila
melanogaster
Chapter 20 slide 88Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
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
Characteristics of Transposable Elements
Some elements have terminal inverted repeats
Characteristics of Transposable Elements
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
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
Mechanism of 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
Transposable 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
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 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
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
Use of TEs as molecular tools
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
Mechanism of transposition
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
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
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
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