1. Transposons
Dr Derakhshandeh

2. Mobile Genetic Elements
Transposons or Transposable elements (TEs)
move around the genome

3. Transposable elements in prokaryotes
Insertion sequence (IS) elements
Transposons (Tn)
Bacteriophage Mu

4. 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)

7. Integration of IS element in chromosomal DNA

8. Three different mechanisms for transposition
Conservative transposition
Replicative transposition
Retrotransposition

9. Conservative transposition: The element itself moves from the donor site into the target site

10. Replicative transposition: The element moves a copy of itself to a new site via a DNA intermediate

11. Retrotransposition: The element makes an RNA copy of itself which is reversed-transcribed into a DNA copy which is then inserted (cDNA)

12. common feature of mobile elements
Generation of short direct repeats flanking the newly inserted element
This results for a staggered cut being made in the DNA strands at the site of insertion

14. Examples of DNA-intermediate mobile elements
Insertion Sequences (IS) elements in bacteria
P elements in Drosophila
AC/DS (dissociation) elements in maize
AC is a full-length autonomous copy
DS is a truncated copy of AC that is non-autonomous, requiring AC in order to transpose
At least seven major classes of DNA transposons in the human genome (3% of total genome)

15. Methods for Generation of Mutant Populations
The most reliable method to ascertain gene function is to disrupt the gene and determine the phenotype change in the resulting mutant individual
Two most popular methods to generate mutants:
1. Insertional mutagenesis
2. Deletional mutagenesis

16. Two main methods
1. Transposon insertion
2. T-DNA insertion

17. All transposable elements fall into one of the following two classes
1. DNA elements
2. Retroelements

18. DNA elements These elements transpose via DNA intermediates such as:
Ac/Ds and Spm in plants, P elements in animals, Tn in bacteria
A common feature of DNA elements is the flanking of the element by short inverted repeat sequences
The enzyme transposase recognizes these sequences, creates a stem/loop structure
excises the loop from the region of the genome
The excised loop can then be inserted into
another region of the genome

19. DNA-Immediate Mobile Genetic Elements
The Short inverted repeats at the ends of the element
These inverted repeats act as the substrates for recombination reactions mediated by the transposase

20. Structure and transposition of a transposable element

21. All transposable elements fall into one of the following two classes
1. DNA elements
2. Retroelements

22. Retroelements transpose via RNA intermediates
The RNA is copied by reverse transcriptase into cDNA
the cDNA integrates into the genome
Retroelements are found in all eukaryotes
such as Tos in rice, copia in animals
Ty1 in yeast

23. Retrotransposon transposition

24. Retorviruses
The basic structure is an LTR = long terminal repeat which flanks three genes,
A complete retroviruses also contains three genes:
gag = structural gene for capsid
Pol = reverse transcriptase
env = envelope gene for the virus

26. How do we use a transposon for mutagenesis?
The insertion and excision of transposable elements
result in changes to the DNA at the transposition site
The transposition can be identified when a known DNA sequence or selection markers are inserted within the elements

27. Transposomics
EZ::TN Transposomes provide an efficient and reliable method for generating a library of random gene knockouts in vivo
Gene inactivation and examination of the resulting phenotype will identify the function of the interrupted genes

28. Transposon-Mediated Homologous Recombination Gene Knockout in Fungi Hamer et al. Proc Natl Acad Sci U S A ;98(9):5110-5

29. T-DNA insertion mutagenesis
T-DNA is a segment of the tumor-inducing (Ti) plasmid of Agrobacterium
delimited by short imperfect repeat border sequences

30. T-DNA transfer from Agrobacterium to plant cell

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

32. Composite transposons

33. IS10R is an autonomous element, while IS10L is non-autonomous

34. 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

35. Composite Transposons
Tetracycline resistance is carried by a Transposable element
The transposon is a composite transposon, composed of IS-element flanking an included sequence
IS10R is an autonomous element
while IS10L is non autonomous
Composite transposons probably evolved from IS elements by the chance location of a pair in close proximity to one another. Inactivation of one element by mutation would not harm ability to transpose and would assure continued transposition of the entire transposon

36. Temperate bacteriophage Mu (Mu = mutator)
37 kb linear DNA with central phage DNA and unequal lengths of host DNA at each end
Mu integrates by transposition
replicates when E. coli replicates
During the lysogenic cycle, Mu remains integrated in E. coli chromosome

37. bacteriophage Mu

38. The advantages / disadvantage of Mu
The advantages of the use of Mu are:
it is not normally found in the bacterial genome
therefore there are few problems with homology to existing sequences in the chromosome; in contrast to most other transposons
Mu does not need a separate vector system
since it is itself a vector
A wide variety of useful mutants of Mu have been generated
The disadvantage of Mu:
it is a bacteriophage and therefore can kill the host cell

40. Drosophila transposons
~15% of Drosophila genome thought to be mobile
2 different classes:
Copia retrotransposons
Conserved, scattered copies/genome
Structurally similar to yeast Ty elements
Use RNA and reverse transcriptase
Eye Color in Drosophila (white apricot wa)

41. DTR
DTR

42. P elements P elements vary in length from 500-2,900 bp
possesses ~40 P elements/genome
Hybrid dysgenesis, defects arise from crossing of specific Drosophila strains
Occurs when haploid genome of male (P strain)
P elements code a repressor, which makes them stable in the P strain in male (but unstable when crossed to the wild type female/; female lacks repressor in cytoplasm)

45. Noncomposite transposons

46. Noncomposite transposons (Tn)
Carry genes (e.g., a gene for antibiotic resistance)
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)

47. Ac (activator)/Ds (dissociation) System discovered by B
Ac (activator)/Ds (dissociation) System discovered by B. McClintock (Noble Prize Winner in 1983)

48. Ac/Ds System

49. Ac/Ds System

50. Schematic Diagram of the Ds Donor Site and Possible Transposition Events

51. Open arrowheads indicate the 5' and 3' ends of th transposon
The Ds element carries the NPTII gene, which confers resistance to kanamycin (KanR)
and a modified GUS reporter gene (Sundaresan
et al )
Possible transposition events include the following:
(1) unlinked or loosely linked transposition to the same chromosome;
(2) transposition to a different chromosome;
(3) closely linked transposition; and
(4) closely linked transposition disrupting theIAAH gene

52. Ac/Ds Transposon tagging system
Advantages: Efficient and cost-effective method to generate a large mutant population
Disadvantages: Secondary transposition complicates gene identification
And transposon system is not available in many species

56. Transposition elements in Human

57. Mobile Genetic Elements and Other Families of Repetitive DNA
The genome is littered with large families of repetitive sequences
have no apparent function in the cell
Mobile Genetic Elements
Tandemly repeated simple sequence DNAs
Satellite DNAs
Short simple repeats (microsatellites)

58. LINEs (Long interspersed elements)
LINEs are one of the most ancient and successful inventions in eukaryotic genomes
In humans, are about 6 kb long
encode two open reading frames (ORFs)
Most LINE-derived repeats are short, with an average size of 900 bp - 1,070 bp
The LINE machinery is believed to be responsible for most reverse transcription in the genome

59. SINEs (Short interspersed elements)
short (about bp)
A single monophyletic family of SINEs (ALU)
This family is the only active SINE in the human genome
The human genome contains three distinct monophyletic families of SINEs: the active Alu, and the inactive MIR and Ther2/MIR3

60. Identification of a human specific Alu insertion in the factor XIII gene
Alu repeats are interspersed repetitive DNA elements specific to primates that are present in 500,000 to 1 million copies
An Alu Insert as the Cause of a Severe Form of Hemophilia A (factor VIII)
Acta Haematol 2001;106:126–129