Seminar helpful for postgraduates. covers all aspects of transposon tagging and mapping.

1. Welcome

2. GENE TAGGING
Gene tagging is referred to assigning some known DNA
sequences in or into the vicinity of a gene of interest
such that the gene can be found with the help of
assigned sequences.
Tag
Gene of Interest

3. Types of Gene Tagging
• Marker based gene tagging
• T-DNA tagging
• Transposon tagging
• Epitope tagging

4. Transposon Mediated Gene
Tagging In Plants

5. TRANSPOSON
These are discrete sequences in the genome that are
mobile and are able to transport themselves within the
genome.
Junk DNA

6. Why these kernels are spotted?

7. Somatic Excision of Ds from C
Wild type Colorless Variegated

8. Types:
• CLASS I or RETROELEMENTS:
Copy and paste mechanism
Move via RNA intermediate
Used for tagging in mammals and yeast
E.g. - Ty1, Copia, Gypsy, LINES, SINES
• CLASS II or DNA-TYPE ELEMETS:
Cut and paste mechanism
Used for tagging in plants, bacteria, drosophila and C. elegans
E.g. – IS elements, Ac/Ds elements, Sleeping beauty, Mu
elements, En/Spm elements

9. TE composition in different crop
plants

10. Transposon tagging
• Transposon tagging describes isolation of novel genes
using transposable elements as tags
• The transposon sequence is used to identify the flanking
sequences after insertional mutagenesis
• The strategy was initially developed to clone the
drosophila white locus (Bingham et al.1981)
• In plants, this strategy was first used to identify and clone
bronze1 (bz1)locus in maize (Federoff et al. 1984)

12. Transposons used
• PLANTS:
• Ac/Ds (Activator/Dissociation) – Maize (Zea mays)
• En/Spm (Enhancer/Suppressor- mutator) – Maize (Zea mays)
• Mu (Mutator) – Maize (Zea mays)
• Tam3 – Snapdragon (Antirrhinum majus)
• ANIMALS:
• Sleeping beauty – Salmon (salmo salar)
• Tol2 – Medaka (Oryzias latipes)

13. APPROCHES:
• TARGETED GENE TAGGING
Tagging of a specific gene for which the mutant is
already available.
• NON TARGETED GENE TAGGING
Tagging any random gene(s) and then studying the
resultant mutant.

14. Directed-Gene Tagging
• Directed tagging identifies the transposon-induced alleles by
crossing transposon active plants with a reference
homozygous mutants.
• The mutable alleles are separated from the reference allele by
crossing to a standard line(hybrid, inbred or tester).
• To identify co-segregating transposon, the mutable allele is
backcrossed to the standard line, and the backcrossed progeny
is selfed.

16. Non-directed Gene Tagging
• In this technique one has to go for M2 population as
compared to F1 in targeted gene tagging.
• The main advantage of this technique is that it can also be
used to study lethal or infertile mutants, ultimately identifying
the gene responsible for it. But targeted tagging is only
restricted to mutants non essential for a plant to complete its
life cycle.

17. • Transposon active stocks are crossed with a standard line and
the resulting progeny is self pollinated.
• The self pollinated progeny is screened for recessive mutants
and segregating populations is generated by crossing with the
standard line for studying co-segregation of transposon along
with mutant phenotype.
• The most common method for co-segregation analysis is
insertion mutagenized sites (AIMS) protocol (Frey et al. 1998)

19. IDENTIFICATION
RFLP followed by SOUTHERN HYBRIDISATION
1. Restriction enzyme employed should be such that it do
not cut within the transposon used.
2. Gel electrophoresis
3. Analysis by southern blotting.
4. Probes are used for the transposon.
RESULTS
 Heterologous system: band present in mutant and
absent in wild type
Endogenous system: bands found in both mutant and
wild type.

20. INVERSE-PCR
1. Ligation of fragment ends
2. Amplifying flanking region of transposon
3. Flanking region used as probe in wild type DNA
CONFIRMATION REQUIRED

21. CONFIRMATION
1. COMPLEMENTATION TEST
The mutant is again transformed with the functional copy of the
gene for which the mutant is expected.
RESULT
Function restored: mutation by tagged gene.
Function not restored: mutation by some other reason.
2. REVERTANTS
The mutant obtained is either selfed (endogenous system) or
crossed with mutant transgenic line with autonomous transposable
element (heterologous system).
RESULT
Revertants obtained: Gene confirmed
Revertants not obtained: mutation by some other reason.

22. Endogenous system

23. Endogenous vs. Heterologous
Endogenous System
• No transformation
experiment needed
• Have high copy no. of
transposon being used
• High rate of mutation is
observed
• Identification of tagged
gene responsible for
mutation is difficult
• E.g. maize, snapdragon
Heterologous system
• Transformation
experiments needed
• Low copy no. of
transposons
• Low rate of mutation is
observed
• Identification of tagged
gene responsible for
mutation is easy
• E.g. potato, tomato

25. Outline:
• Cf-9 gene was known to provide resistance against leaf mould
fungus Cladosporium fulvum in tomato
• Cf-9 gene was already mapped on short arm of chromosome 1
• Avr9 genes were characterised in race 5 of fungus C. fulvum
• Plant defenses are often activated by specific interaction
between the product of a disease resistance (R) gene in the
plant and the product of a corresponding avirulence (Avr)
gene in the pathogen. Without either of these genes, plant
defenses are not activated and infection by the pathogen is
permitted.

26. Materials:
• A transgenic tomato line carrying a Ds element located 3
centimorgans from the Cf-9 locus
• A stable line containing genetically unlinked Ac (sAc), itself
incapable of transposition
• A line homozygous for Cf-9
• A line homozygous for Avr9

28. • A total of at least 37 independent Ds insertions into Cf-9. Of
these, 28 have been mapped to the same 3-kb region of the
tomato genome
• All stable mutants were susceptible to race 5 of C. fulvum
• Correlation between multiple independent mutations of Cf-9
and multiple independent Ds insertions in a defined region
• In plants which carried sAc in heterozygous state, they found
revertants, thus confirming the gene tagged was correct.

30. • Y1 allele is responsible for yellow endosperm color while y1
result in white color.
• They used Mu3 transposon to tag Y1 allele

32. Revertants

34. Conclusion
• Initially only transposon sequence is required
• Used to identify and clone numerous genes having visible
phenotypes.
• Aid in mutation studies
• Any specific as well as random genes can be targeted by this
method
• Can be used to produce an allelic series of a gene.

36. THANK YOU