Introduction of transposons, structure, mechanism and application of transposons

1. Presented By:- Ms. Hemali Kachhadiya
Assistant Professor,
Department of Microbiology
Swarrnim Science College

2. Transposons (Transposable Genetic Elements) are pieces of DNA that can move
from one location on the chromosome another, from plasmid to chromosome or vice
versa or from one plasmid to another.
Portions change locations within the chromosome, or they may copy into a new
location.
Transposable elements were first discovered by Barbara McClintock in the 1950's
Transposase is the enzyme that carries out the transposition process.

3. • Transposons were "re-discovered" in bacteria in the 70's as "insertion
sequences", genetic elements which move from one genetic location to
another and that cause premature transcriptional termination in
bacteria operons
• Bacterial transposons are common and varied. Normal E. coli strains
carry more than one copy of transposons IS1, IS2, IS3, and IS4

4. • The smaller transposons such as the IS elements do not encode additional functions to
transposition. Larger transposons often carry genes that confer selective advantage to the
transposon-carrying cell: resistance to antibiotics is most common, resistance to Hg,
toxin production and fermentation genes are also found.
• Larger transposons are often composite transposons. That is, they are made up of IS
elements flanking a drug-resistance gene.
• The IS elements are capable of independent transposition without carrying drug-
resistance.
• Transposons play an important role in the evolution of bacterial genomes by providing a
mechanism for inheritance of genes via nonhomologous recombination.
• In this way transposable elements also facilitate horizontal gene transfer between
unrelated organisms.

5. • Approximately 15% of the human genome is believed to be composed of
transposable elements. Most eukaryotic transposons are of the retrotransposon
type but some DNA elements are also found.
• Examples include P elements from Drosophila which have been used extensively
for gene transfer and have been well-studied; also the Ac/Ds elements originally
discovered by McClintock in maize.

6.  Simple transposition
 Replicon fusion
 Adjacent deletion
 Adjacent inversion

7. 1. Inverted repeat at ends
2. Direct repeat generated in target 5-13 bp.
3. Transposase gene tnp

8. 3 types based on the mechanism which is used for transposition:
1. Replicative or co-integrate-forming transposons
2. Conservative (non-replicative) transposons
3. Retro-transposons

9. 3 genes: tnpA, tnpR, res.
1. tnpA: Transposition-negative.
2. tnpR: Elevated levels of transposition (acts genetically like repressor).
Co-integrate intermediate accumulate.
3. res. ICs-acting. Generally like tnpR phenotype.
 Modified Shapiro model of transposition explains:
1. Direct repeat in target.
2. Obligate co-integrate intermediate.
3. Dependence on replication.
4. Side reaction without replication (as seen at low freq. in tnpR mutants) give
conservative type.

10. Suicide-donor model. Repair can occurs by homologous recombination with
homolog or sister.
1. Explains why no tnpR-type gene needed.
2. Predicts low level of co-integrant in general but could be produced.
3. Kleckner experiment with heteroduplex transposon.

11. • Retrotransposons represent a highly unique group of transposable
elements and form large portions of the genomes of
many eukaryotes (organisms with cells containing a clearly
defined nucleus). Retrotransposons function by a “copy and paste”
mechanism.
• Thus, they leave behind the original copy and generate a second copy
that is inserted elsewhere in the genome.
• This process results in the insertion of repetitive sequences of DNA
throughout the genome and is the mechanism responsible for the vast
spread of transposable elements in many higher organisms.

12. 1. Excessive transposition would kill cell. Only high frequency
transposon is Mu, which does kill cell.
2. Low levels of transposition are probably because transposase is
poorly expressed:
3. Transposition definitely higher in "virgin" cells.
4. Stress: probably not. However, in some cases, transposition is much
higher at low temperatures.

14. Structure of a Transposon:
• Transposons are stretches of DNA that have repeated DNA segments
at either end. A transposon consists of a central sequence that has
transposes gene and additional genes. This is flanked on both sides by
short repeated DNA segments. The repeated segments may be direct
repeats or inverted repeats. These terminal repeats help in identifying
transposons.
• The number of repeated nucleotides is uneven 5 or 7 or 9 nucleotides
are due to its method of insertion at the target site.

15. Mechanism of Transposition
• The enzyme transposase present
in the transposon itself makes
nicks or cuts in each strand of the
target sequence. The target
sequence is duplicated and two
copies move away to make way
for the transposon in the centre.
• The transposon then fixes itself
into the two free ends generated.
The nicks are sealed by ligase and
two strands become continuous.

16. Types of Transposons
• On the basis of their transposition mechanism, transposons may be
categorized into following types:
1. Cut-and-Paste Transposons
2.Replicative Transposons
3.Retro Elements

17. Cut-and-Paste Transposons
• They transpose by excision (cutting) of the transposable sequence
from one position in the genome and its insertion (pasting) to another
position within the genome (Figure)
The cut-and-paste transposition involves two transposase subunits. Each
transposase submit binds to the specific sequences at the two ends of
transposon. These subunits of transposase protein then come together and
lead to the excision of transposon.

18. • This excised ‘transposon-
Transposase Complex’ then gets
integrated to the target recipient
site. In this manner, the transposon
is cut from one site and then
pasted on other site by a
mechanism mediated by
transposase protein (Figure).
• Examples of cut-and-paste type of
transposons are IS-elements, P-
elements in maize, hobo-elements
in Drosophila etc.
Cut-and-Paste Transposons

19. Replicative Transposons
• they transpose by a mechanism which involves replication of
transposable sequence and this copy of DNA, so formed, is inserted
into the target site while the donor site remains unchanged (Fig. 3).
Thus, in this type of transposition, there is a gain of one copy of
transposon and both-the donor and the recipient DNA molecule are
having one-one transposable sequence each, after transposition.
• Tn3-elements found in bacteria are good examples of such type of
transposons.

20. Retro Elements
• Their transposition is accomplished through a process which involves
the synthesis of DNA by reverse transcription (i.e. RNA DNA) by
using elements RNA as the template (Figure). This type of
transposition involves an RNA intermediate, the transposable DNA is
transcribed to produce an RNA molecule.

21. Retro Elements
• This RNA is then used as a template for producing a complementary
DNA by the activity of enzyme reverse transcriptase. This single
stranded DNA copy so formed, is then made double stranded and then
inserted into the target DNA site. The transposable elements which
require reverse transcriptase tor their movement are called retro
transposons.
• The Retro elements may be viral or non-viral. Out of these two, the
non-viral retro elements are important and may further be classified as:
1. Retrovirus like elements
2. Non-LTR Retrotransposons

22. 1) Retrovirus like elements
• They carry long terminal repeats (LTR). Examples are copia, gypsy
elements in Drosophila.
2) Non-LTR Retrotransposons
• LTR are absent. Examples are LINEs and SINEs in humans
Retro Elements

24. Use of Transposons
• As cloning vehicles.
• Transformation vectors for transferring genes between organisms.
• Also drug resistance genes encoded by many transposons are useful in
the development of plasmids as cloning vehicles.
• Transposons mutagenesis.
• Use of transposons to increase rate of mutation due to insertional
inactivation.

25. Transposons as genetic markers
• Transposons may be used as genetic markers because they change the
pattern of restriction fragment analysis (i.e. the results of analysis of
DNA fragments obtained after digestion with specific restriction
endonucleases).
• Transposons, which change the pattern of restriction fragment
analysis, may also be used as genetic markers to construct linkage
maps. These have also been used in humans for distinguishing carriers
from non-carriers of disease like sickle cell trait. A set of cloned DNA
fragments containing copies of transposons, should also contain
unique sequences from specific regions of the genome and can be used
in such studies.

26. Transposons as mutagens
• Insertion of transposons can be used as a method for inducing
mutations as has been shown in a number of spontaneous mutations
like Ac-Ds system in maize, and P-M and I-R systems of hybrid
dysgenesis in Drosophila melanogaster.
• Transposons usually cause mutations due to insertion in structural or
regulatory region, rather than due to addition, deletion or substitution
of bases. Therefore, these mutations can be used for a study of
structural and regulatory regions of a gene.

27. Transposon tagging for isolation of
genes
• A probe carrying a transposon can be used to screen the restriction
fragments containing the mutant gene. This feature of transposons has
also been utilized for isolation of genes though transposon tagging.

28. Transposons as transformation
vectors
• genes can be transferred artificially if carried by a transposon in
a plasmid.
• Transposons of higher plants (e.g. Ds, Ac or Mul of maize) and
transposons of Drosophila (P elements) are also used as
vectors
• at present the commonly used vector for higher plants is only Ti
plasmid of Agrobacterium tumefaciens, which can be used
conveniently only with dicotyledonous plants.