This document summarizes transposable elements and transposon mutagenesis. It discusses the general features of transposable elements, including their mechanisms of movement and ability to cause genetic changes. It then describes various types of transposable elements found in prokaryotes and eukaryotes. In prokaryotes, examples discussed include insertion sequences, transposons like Tn10, and bacteriophage Mu. In eukaryotes, examples of plant transposons like Ac-Ds, yeast retrotransposons like Ty, and Drosophila transposons like copia and P elements are summarized. The structures, mechanisms of movement, and effects of insertion of several of these transposable elements

1. Chapter 20 slide 1
Transposable Elements and
Transposon mutagenesis
Govt. College Bundi
Presented by
Simran
M.sc. Botany sem.III

2. Chapter 20 slide 2
General Features of Transposable Elements
1. 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.

3. Chapter 20 slide 3
Transposable Elements in Prokaryotes
1.Prokaryotic examples include:
a.Insertion sequence (IS) elements.
b.Transposons (Tn).
c. Bacteriophage Mu (replicated by transposition)

4. Chapter 20 slide 4
Insertion Sequences
Animation: 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.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).

5. Chapter 20 slide 5
Fig.1 The insertion sequence (IS) transposable element, IS1

6. Chapter 20 slide 6
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 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

7. Chapter 20 slide 7
Fig.2 Schematic of the integration of an IS element into chromosomal DNA

8. Chapter 20 slide 8
Transposons
1. 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 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).

9. Chapter 20 slide 9
Fig. 3 Structure of the composite transposon Tn10

10. Chapter 20 slide 10

11. Chapter 20 slide 11
Fig. 4 Structure of the noncomposite transposon Tn3

12. Chapter 20 slide 12
Fig.5 DNA sequence of a target site of Tn3

13. Chapter 20 slide 13
2. Models have been generated for transposition:
a. Cointegration is an example of the replicative transposition that
occurs with Tn3 and its relatives (Figure 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.

14. Chapter 20 slide 14
Fig. 6 Cointegration model for transposition of a transposable element by
replicative transposition

15. Chapter 20 slide 15
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 7).
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 8).

16. Chapter 20 slide 16
Fig.7 Temperate bacteriophage Mu genome shown in (a) as in phage particles and
(b) as integrated into the E. coli chromosome as a prophage

17. Chapter 20 slide 17
Fig. 8 Production of deletion or inversion by homologous recombination between
two Mu genomes or two transposons

18. Chapter 20 slide 18
Transposable Elements in Eukaryotes

19. Chapter 20 slide 19
Transposons in Plants
Animation: 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).

20. Chapter 20 slide 20
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 9).
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.
Fig.9 purple pigment on
white kernels
Barbara McClintock

21. Chapter 20 slide 21
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 10).
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.

22. Chapter 20 slide 22
Fig.10 Kernel color in corn and transposon effects

23. Chapter 20 slide 23

24. Chapter 20 slide 24
Fig. 11 The structure of the Ac autonomous transposable element of corn and of
several Ds nonautonomous elements derived from Ac

25. Chapter 20 slide 25
Fig. 12 The Ac transposition mechanism

26. Chapter 20 slide 26
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.

27. Chapter 20 slide 27
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 13:
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 13).
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.

28. Chapter 20 slide 28
Fig. 13 The Ty transposable element of yeast

29. Chapter 20 slide 29
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 14).
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).

30. Chapter 20 slide 30
Fig. 14 Structure of the transposable element copia, a retrotransposon found in
Drosophila melanogaster

31. Chapter 20 slide 31
b. P elements cause hybrid dysgenesis, a series of defects
(mutations, chromosomal aberrations and sterility)
that result from crossing certain Drosophila strains
(Figure 15).
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.

32. Chapter 20 slide 32
Fig. 15 Hybrid dysgenesis, exemplified by the production of sterile flies

33. Chapter 20 slide 33
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 17):
(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.

34. Chapter 20 slide 34
Fig. 16 Structure of the autonomous P transposable element found in Drosophila
melanogaster

35. Chapter 20 slide 35
Fig. 17 Illustration of the use of P elements to introduce genes into the Drosophila
genome

36. Chapter 20 slide 36
Human Retrotransposons
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.

37. Chapter 20 slide 37
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.)

38. 1.Transposon mutagenesis is a biological process that
allows genes to be transferred to a host organism's
chromosome, interrupting or modifying the function of
gene on the chromosome and causing mutation(figure
18).
2.It is much more effective than chemical mutagenesis,
with a higher frequency and a lower chance of killing the
organism.
Transposon MutagenesiS

39. K A Ntr
EZ:: TN Transposon
Not l Not l
Target
clone
EZ:: TN Transposase
1. Incubate 37⁰C ; 2 hrs
2.Transform E.coli
3.select KAN clones
R
4. Map or sequence insertion sites (optional)
5. Digestion with NOT l. Religate to generate 19- codon insertion Transform E.coli.
6.Express the protein.,Assay for mutations,altered activity, etc.
Not l Not l Not l
COOH COOH COOH
NH3 NH3 NH3
Fig.18 Transposon mediated mutagenesis

40. TRANSPOSON AS TOOLS FOR MUTAGENESIS
A transposon used for mutagenesis should
have following properties:
i.It should transpose at a fairly high frequency
ii. It should not be very selective in its target
sequence
iii. It should carry an easily selectable gene,
such as one for resistance and one for
antibiotic.
iv. Should have broad host range for
transposition

41. TRANSPOSON MUTAGENESIS IN VIVO
1.Transposon Tn5 is ideal for random mutagenesis
of gram negative bacteria as it embodies all of its
features.
2.Tn5 transpose at a relatively high frequency but
has no target specificity.
3.It also carries a kanamycin resistance gene that
is expressed in most gram negative bacteria.
ADVANTAGE:
The target organism does not have to be naturally
competent.

42. 1.The transposon must be introduced into the host on a
suicide vector, which may give some residual false positive
results for transposon insertion mutants if the suicide
vector is capable of limited replication.
2.It is not very effective and requires powerful positive
selection techniques to isolate the mutants.
3.If DNA sequence or specific plasmid is to be mutated,
there is no target specificity to insertion mutants so
transposon hops into chromosome most of the time. There
is also possibilty of multiple transposon events.
DISADVANTAGE
:

43. TRANSPOSON MUTAGENESIS IN VITRO
1.This technology is made possible by the fact that
the transposase enzyme by itself performs the
reactions of "cut and paste phase" transposition
reaction.
2.In the procedure the target DNA is mixed with a
donor DNA containing the trasnposon, and the
purified tansposase is added allowing the
transposon to insert into the target DNA in the test
tube(figure 19).
3.Multiple transposases have been adapted for this
process.

44. PCR amplification cloning into
binary vector (R&L Gateway )
Random transposon
mutagenesis
Transposon
Transformation screening for plasmid with
selection marker inserted into the HRS
Finished vector
Fig.19 In vitro transposon mutagenesis

45. ADVANTAGES:
It has the ability to reach high saturation levels of
mutagenesis, which allows one to conduct annalysis of the
target locus on either large or small scales.
DISADVANTAGE:
It has the prerequisite for preliminary information on
the target sequence.

46. 1.Virulence genes in viruses and bacteria can be
discovered by disrupting genes and observing for a
change in phenotype.
2. Non essential genes can be discovered by
inducing transposon mutagenesis in an organism
with the help of PCR and ORF specific primer.
3.Cancer causing genes can be identified by
transposon mutagenesis and screening of mutants
containing tumours.

47. SITE DIRECTED MUTAGENESIS

48. S
M
urs S'
M' P
PCR
Purification and digestion
Ligation, digestion and transformation
Fig.20 Schematic representation of site directed mutagenesis

49. DIFFERENT TECHNIQUES

50. A. CONVENTIONAL PCR
1. In this method PCR primers are designed in a
manner which contains mutation.
2. The Taq DNA polymerase used in the conventional
PCR does not have exonuclease activity hence it
cannot identify mismatch during the amplification
3. The major reccomendation for the conventional
PCR based mutagenesis is to insert mutant bases up
to several limit at 5' end of the primer or in the
middle of the primer.
4. LIMITATION: It carries mutant as well as non

51. B. PRIMER EXTENSION/NESTED PCR
1. Two sets of primers are used in which a single set of
primer is nested.
2. The mutation is introduced to the primer at one end.
C. INVERSE PCR
1.The primers amplify the fragment other than the target
sequence, hence it amplifies in the reverse orientation.
2. The method is used for inserting mutation into the plasmid
having the gene of our interest.
3. The fidelity DNA polymerase is used to do the
amplification as well as to linearise the circular plasmid
DNA
4. Many nucleotides can be deleted by using inverse
PCR(figure 21).

52. Intermolecular ligation
Linear DNA molecules
Inverse PCR
Portion of DNA deleted
Fig.21 Deletion by inverse PCR

53. 1. The site directed mutagenesis is used to remove
restriction sites.
2. Restriction digestion is a process in which the
DNA having the recognition site for a particular
restriction endonuclease is cleaved into fragments.
3. If any mutation is introduced at the recognition
site of REase, it cannot cut it.This can be done by site
directed mutagenesis.
4. At molecular level, the properties of a molecular
gene or proteins can be screened or studied with the
IMPORTANCE

54. APPLICATIONS
1. SDM helps to improve the quality of protein by
removing harmful elements from it.
2. The tool is used in study of a gene characteristics.
3. Used in gene synthesis and gene editing technology.
4. Used in cloning.
5. It is also useful in the screening of single nucleotide
polymorphisms(SNPs).

55. CONCLUSION
1. SDM has its own importance in the field of
gene editing and gene manipulation
2. It facilitates improvement in the wild type
genotype to produce a commercially important
phenotype
3. SDM has employed in the knockout mice
construction and gene knockout studies.

56. Thank You