1. Transposons
Dr Gihan Gawish

2. Transposable element
genetic elements of a chromosome that have the capacity to mobilize and move from one location to another in the genome.

3. Discovery of Transposable Elements
Barbara McClintock
( )
Her genetic studies in the 40’s and 50’s of spotted kernels in maize and of the cytogenetics of broken chromosomes led her to the discovery of the “controlling elements” Dissociation (Ds) and Activator (Ac)

4. The transposition first discovered in 1948 by Barbara McClintock was initially thought to be unique to maize (corn) but later recognized to be common in eukaryotes, bacteria, viruses, phages and plasmids (a Nobel Prize in 1983).

5. Transposons are segments of DNA that can move around to different positions in the genome of a single cell. In the process, they may •cause mutations •increase (or decrease) the amount of DNA in the genome

6. These mobile segments of DNA are sometimes called "jumping genes".

7. There are two distinct types of transposons:
• DNA transposons - transposons consisting only of DNA that moves directly from place to place.
(prokaryotes& eukaryotes).
Retrotransposons that
~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.
(eukaryotes only).

8. There are two classes of transposons.
1. Retrotransposons are transcribed to RNA and back to DNA by reverse transcriptase. The DNA copy is pasted into multiple positions of genome. This increases genome size.
2. DNA transposons are cut and pasted into other positions of genome by a transposase.
Some DNA transposons replicate themselves at new sites (replicative transposition).

9. In both cases dsDNA intermediate is integrated into the target site in DNA to complete movement

10. Transposition
Many non‐coding regions of DNA are repetitive such as transposons (transposable elements, or mobile genetic elements) 'selfish DNA‘
Some transposons move without replication

11. Transposable elements in prokaryotes:
Insertion sequence (IS) elements
Transposons (Tn)

12. Insertion sequence (IS) elements:
Simplest type of transposable element found in bacterial chromosomes and plasmids.
Encode only genes for mobilization and insertion.
Range in size from 768 bp to 5 kb.
IS1 first identified in E. coli’s glactose operon is 768 bp long and is present with 4-19 copies in the E. coli chromosome.
Ends of all known IS elements show inverted terminal repeats (ITRs).

15. Insertion sequence (IS) elements:
Integration of an IS element may:
Disrupt coding sequences or regulatory regions.
Alter expression of nearby genes.
Cause deletions and inversions in adjacent DNA.
Result in crossing-over.

16. Transposition of insertion sequence (IS) elements:
Original copy remains in place; new copy inserts randomly.
IS element uses host replication enzymes for replication.
Transposition requires transposase, coded by the IS element.
Transposition initiates when transposase recognizes ITRs.
Site of integration = target site.
Staggered cuts are made in DNA at target site, IS element inserts, DNA polymerase and ligase fill the gaps.
Small direct repeats (~5 bp) flanking the target site are created.

17. Integration of IS element in chromosomal DNA.

18. Transposons (Tn):
Similar to IS elements but are more complex structurally and carry additional genes
2 types of transposons:
Composite transposons
Noncomposite transposons

19. Composite transposons (Tn):
Carry genes (e.g., a gene for antibiotic resistance) flanked on both sides by IS elements.
Tn10 is 9.3 kb and includes 6.5 kb of central DNA (includes a gene for tetracycline resistance) and 1.4 kb inverted IS elements.
IS elements supply transposase and ITR recognition signals.

21. Noncomposite transposons (Tn):
Carry genes (e.g., a gene for antibiotic resistance) but do not terminate with IS elements.
Ends are non-IS element repeated sequences.
Tn3 is 5 kb with 38-bp ITRs and includes 3 genes; bla (-lactamase), tnpA (transposase), and tnpB (resolvase, which functions in recombination).
Fig. 7.21b

22. Models of transposition:
Similar to that of IS elements; duplication at target sites occurs.
Cointegration = movement of a transposon from one genome (e.g., plasmid) to another (e.g., chromosome) integrates transposon to both genomes (duplication).
May be replicative (duplication) or non-replicative (transposon lost from original site).
Result in same types of mutations as IS elements: insertions, deletions, changes in gene expression, or duplication.
Crossing-over occurs when donor DNA with transposable element fuses with recipient DNA.

23. Recombination, crossing-over, and duplication of a transposable element.

24. Transposable elements in eukaryotes:
Barbara McClintock ( )
Cold Spring Harbor Laboratory, NY
Nobel Prize in Physiology and Medicine 1983
“for her discovery of mobil genetic elements”
Studied transposable elements in corn (Zea mays) 1940s-1950s
(formerly identified as mutator genes by Marcus Rhoades 1930s)

25. Transposons in Eukaryote
Eukaryotic genomes have many copies of transposons (∼45% of the human genome).
Transposition accompanies insertion in a genome site and some (not all) insertions can cause changes in gene expression, its regulation and products and can lead to speciation or diseases.
The insertion sites are random, although some sites (called hot spots) are preferred to others

26. General properties of plant transposons:
Possess ITR sequences and generate short repeats at target sites.
May activate or repress target genes, cause chromosome mutations, and disrupt genes.
Two types:
Autonomous elements transpose themselves; possess transposition gene.
Nonautonomous elements do not transpose themselves; lack transposition gene and reply on presence of another Tn
McClintock demonstrated purple spots in otherwise white corn (Zea mays) kernels are results of transposons.

27. McClintock’s discovery of transposons in corn:
Purple spots in white corn kernels are results of transposons.
c/c = white kernels and C/- = purple kernels
Kernal color alleles/traits are “unstable”.
If reversion of c to C occurs in a cell, cell will produce purple pigment and a spot.
Earlier in development reversion occurs, the larger the spot.
McClintock concluded “c” allele results from a non-autonomous transposon called “Ds” inserted into the “C” gene (Ds = dissassociation).
Autonomous transposon “Ac” controls “Ds” transposon (Ac = activator) .

28. Transposon effects on corn kernel color.

29. McClintock’s discovery of transposons in corn (cont.):
Ac element is autonomous/Ds element in nonautonomous.
Ac is 4,563 bp with 11 bp ITRs and 1 transcription unit encoding an 807 amino acid transposase.
Ac activates Ds; Ds varies in length and sequence, but possesses same ITRs as Ac.
Many Ds elements are deleted or rearranged version of Ac; Ds element derived from Ac.
Ac/Ds are developmentally regulated; Ac/Ds transpose only during chromosome replication and do not leave copies behind.

30. Structure of Ac autonomous and Ds non-autonomous transposable elements in corn.

31. Ac transposition mechanism during chromosome replication.

32. Ty elements in yeast:
Similar to bacterial transposons; terminal repeated sequences, integrate at non-homologous sites, with target site duplication.
Ty elements share properties with retroviruses, retrotransposons:
Synthesize RNA copy and make DNA using reverse transcriptase.
cDNA integrates at a new chromosomal site.

34. Drosophila transposons:
~15% of Drosophila genome thought to be mobile.
P elements
Hybrid dysgenesis, defects arise from crossing of specific Drosophila strains.
Occurs when haploid genome of male (P strain) possesses ~40 P elements/genome.
P elements vary in length from 500-2,900 bp.
P elements code a repressor, which makes them stable in the P strain (but unstable when crossed to the wild type female; female lacks repressor in cytoplasm).
Also used experimentally as transformation vectors.

36. Female
P elements
DNA + cytoplasm
Repressor
Stable
Female
No P elements
DNA + cytoplasm
No repressor
Male
P elements
DNA only
Male
No P elements
DNA only
Offspring
P elements
No repressor
Unstable germ line

37. Illustration of the use of P elements to introduce genes into the Drosophila genome

38. Human retrotransposons:
Alu1 SINEs (short-interspersed sequences)
~300 bp long, repeated 300, ,000X.
Flanked by 7-20 bp direct repeats.
Some are transcribed, thought to move by RNA intermediate.
AluI SINEs detected in neurofibromatosis (OMIM ) intron; results in loss of an exon and non-functional protein.
L-1 LINEs (long-interspersed sequences)
6.5 kb element, repeated 50, ,000X (~5% of genome).
Contain ORFs with homology to reverse transcriptases; lacks LTRs.
Some cases of hemophilia (OMIM ) known to result newly transposed L1 insertions.

39. The movement of a transposable element can generate mutations or chromosomal rearrangements and thus affect the expression of other genes

40. References Lodish et al., Molecular Cell Biology, 5th edition.
T. A. Brown, Genomes, 1999, Wiley-Liss, New-York.
Nature Reviews Molecular Cell Biology 2; (2001).
Weaver, Molecular Biology, 2005, McGraw Hill.
Peterson-Burch, B.D., Wright, D.A., Laten, H.M. &.Voytas, D.F “Retroviruses in Plants?” Trends Genet. 16: