1. Transposons & Mechanisms of Transposition
Donnie Pickel, Elizabeth Jensen and Dasmeet Kaur

2. Mobile DNA element or transposable element
Interspaced repeats have the capability to “move around” in the genome
“jumping genes”
“selfish DNA”-- exist only to maintain themselves ?
“junk DNA”-- ???
Transposition -- The process by which these sequences are copied and inserted into a new site in the genome
Significant role in evolution -- Positive and negative impacts on genome
*gene inactivation
*modulate gene expression
*induce illegitimate recombination
Selective transposition in germ cells are passed down to successive generations for their accumulation in the genome.
~25% of human promoter regions and ~ 4% of human exons derived

3. Late 1960s, transposition was also found in Bacteria.
1940s, Barbara McClintock discovered the first transposable element in maize
Eventually earned vindication when she became the only woman to independently win the Nobel Prize in medicine (1983).
Late 1960s, transposition was also found in Bacteria.

4. DNA Transposons and Retrotransposons
Transposons excise themselves and reintegrate elsewhere
Retrotransposons follow replicative mechanism and reintegrate themselves

5. Most mobile elements in bacteria is DNA transposons
Mobile elements in eukaryotes are dominated as retrotransposons than eukaryotic DNA transposons.
The relative amount of retrotransposons and DNA transposons in diverse eukaryotic genomes
(Sc: Saccharomyces cerevisiae; Sp: Schizosaccharomyces pombe; Hs: Homo sapiens; Mm: Mus musculus; Os: Oryza sativa; Ce: Caenorhabditis elegans; Dm: Drosophila melanogaster; Ag: Anopheles gambiae, malaria mosquito; Aa: Aedes aegypti, yellow fever mosquito; Eh: Entamoeba histolytica; Ei: Entamoeba invadens; Tv: Trichomonas vaginalis.)
Feschotte, C. & Pritham, E. J. (2007) Annual Reviews in Genetics

6. DNA transposons
Retrotransposons

7. “cut-and-paste” mechanism Mostly “copy-and-paste” mechanism
Lodish et al., Molecular Cell Biology, 7th ed. Fig 10-8
Mostly
“cut-and-paste” mechanism
Mostly
“copy-and-paste” mechanism

8. Classes of Transposable elements

9. IS: Insertion Sequence
Bacterial Insertion Sequences (IS element)
General structure of bacterial IS elements
Lodish et al., Molecular Cell Biology, 7th ed. Fig 10-9

10. Retrotransposons LTR retrotransposons
Non-LTR retrotransposons - transposons in mammals generally
General structure of eukaryotic LTR retrotransposons
Lodish et al., Molecular Cell Biology, 7th ed. Fig 10-11

11. Autonomous & Nonautonomous
Autonomous (activator elements) are similar in structure and function to bacterial IS elements
example-Ac elements
Nonautonomous (dissociation elements) lack transposase gene and cannot move by themselves
-Must have a cis transposable element with transposase gene to move
example-Miniature Inverted-repeat Transposable Elements (MITEs)
-Ds elements

12. DNA transposons families
-depending on their sequence, TIRs, and/or TSDs:
Subclass I: Tc1/mariner
PIF/Harbinger
hAT
piggyBac  
Subclass II: Helitron 
Maverick

13. Mechanisms of Transposition
Lodish et al., Molecular Cell Biology, 7th ed. Fig 10-8

14. Diversity of Transpositions
These two slides are just to show the vast diversity of transposons and that I will be focusing on only a particular few – like Tc/Mariner, IS elements, and briefly the retrotransposons, like LTR and LINE

15. DDE Transposons
More diversity

16. Lodish et al., Molecular Cell Biology, 7th ed. Fig 10-8
DNA Transposons

17. Bacterial IS Element Transposition
Steps (E. coli):
Precise excision of IS element from donor DNA
Staggered cuts in short sequence in target DNA
Ligation
DNA polymerase fills in the single stranded gaps
DNA ligase joins the free ends
Lodish, et al. Molecular Cell Biology, Fig. 10-9

18. Tc1/mariner

19. Retrotransposons Types: Long Terminal Repeats (LTR) Retrotransposons
Lodish et al., Molecular Cell Biology, 7th ed. Fig 10-8
Retrotransposons
Types:
Long Terminal Repeats (LTR) Retrotransposons
Non-LTR Retrotransposons
LINES
SINES

20. LTR Retrotransposons
RNA intermediate transcribed from the mobile element by RNA polymerase
Reverse transcription to convert the RNA into double stranded DNA by reverse transcriptase
Like Retroviruses

21. Non-LTR Retrotransposition (LINE)
Lodish, et al. Molecular Cell Biology, Fig

22. Transposon-Seq (Tn-Seq)
Essential Gene Identification
Genetic Interaction Discovery
Genetic Factors in Biological Processes
Regulatory Factor Discovery

23. Tn-Seq Method
Image from Chao et al. 2016

24. Transposons in Gene Therapy
Lower Production Costs
Increased Biosafety
Low Immunogenecity

25. Mechanisms
Image from Woodard and Wilson, 2015

26. Sleeping Beauty Piggybac Tc1/Mariner-like Elements
Relatively Small Carrier (6-18kb)
Limiting Conc. Of Transposon
Overproduction Inhibition
TA Dinucleotides (random)
Low Promoter/Enhancer Activity
Better Transgenesis Efficiency
Tc1/Mariner-like Elements
Relatively Large Carrier (14-100kb)
TTAA Incoporation Sites
3-5 Nucleotide Mobilization Footprint
Overproduction Inhibition
Transient Expression
Inducible Vector
Non-Specific Incorporation
Preferential Incorporation at Transcription Start Sites

27. References
Feschotte, C. & Pritham, E. J. (2007) DNA transposons and the evolution of eukaryotic genomes. Annual Reviews in Genetics 41, 331–348.
Lodish et al., Molecular Cell Biology, 7th ed.
Pray, L. (2008) Transposons: The jumping genes. Nature Education 1(1):204
López, M.M. and García-Pérez, J.L. (2010) DNA Transposons: Nature and Applications in Genomics Curr Genomics 11(2): 115–128. doi:   /
Curcio MJ and Derbyshire, KM. (2003). The outs and ins of transposition: from Mu to Kangaroo. Nature Reviews Molecular Cell Biology, 4,
Morris, ER et al. (2016) A bend, flip and trap mechanism for transposon integration. eLIFE; 5:e DOI: /eLife
Munoz-Lopez M. and Garcia-Perez JL. (2010). DNA Transposons: Nature and Applications in Genomics. Current Genomics, 11,
Chao, M. C., Abel, S., Davis, B. M., & Waldor, M. K. (2016). The design and analysis of transposon insertion sequencing experiments. Nature Reviews: Microbiology,
Kwon, Y. M., Ricke, S. C., & Mandal, R. K. (2016). Transposon sequencing: methods and expanding applications. Applications of Microbiology in Biotechnology,
Vargas, J. E., Chicaybam, L., Stein, R. T., Tanuri, A., Delgado-Canedo, A., & Bonamino, M. H. (2016). Retroviral vectors and transposons for stable gene therapy: advances, current challenges, and perspectives. Journal of Translational Medicine.
Woodard, L. E., & Wilson, M. H. (2015). piggyBac-ing models and new therapeutic strategies. Trends in Biotechnology,