Transposons

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)

6. Integration of IS element in chromosomal DNA

7. Three different mechanisms for transposition • Conservative transposition • Replicative transposition • Retrotransposition

8. Three different mechanisms for transposition • Conservative transposition: The element itself moves from the donor site into the target site • Replicative transposition: The element moves a copy of itself to a new site via a DNA intermediate • Retrotransposition: The element makes an RNA copy of itself which is reversed-transcribed into a DNA copy which is then inserted (cDNA)

9. Conservative transposition

10. Replicative transposition

11. Retrotransposition

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

15. Composite transposons

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

17. Composite Transposons • Tetracycline resistance is carried by a transposable element • The transposon is a composite transposon, composed of IS-elements flanking an included sequence, in this case containing an antibiotic resistance gene • 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

18. Noncomposite transposons

19. 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)

20. 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)

21. 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. Deletion mutagenesis

22. Two main methods • 1. Transposon insertion • 2. T-DNA insertion

23. Transposon mutagenesis • Transposable elements or transposons • sections of DNA (sequence elements) • move, or transpose, from one site in the genome to another

24. All transposable elements fall into one ofthe following two classes • 1. DNA elements • 2. Retroelements

25. 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

26. 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

27. Structure and transposition of a transposable element

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

29. Retrotransposon transposition

30. 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

32. How do we use a transposonfor 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

33. 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

34. Transposon-Mediated Homologous RecombinationGene Knockout in FungiHamer et al. 2001. Proc Natl Acad Sci U S A. 2001 24;98(9):5110-5

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

36. T-DNA transfer from Agrobacterium to plant cell

37. 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

38. bacteriophage Mu

39. The advantages / disadvantage of Mu • The advantagesof 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

41. Drosophila transposons • ~15% of Drosophila genome thought to be mobile • 2 different classes: • Copia retrotransposons • Conserved, 5-100 scattered copies/genome • Structurally similar to yeast Ty elements • Use RNA and reverse transcriptase • Eye Colorin Drosophila (white apricot wa)

42. ITR(17bp) ITR(17bp) DTR DTR

43. 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 in male (but unstable when crossed to the wild type female/; female lacks repressor in cytoplasm)

46. Ac (activator)/Ds (dissociation)Systemdiscovered by B. McClintock (Noble Prize Winner in 1983)

47. Ac/Ds System

48. Ac/Ds System

49. Schematic Diagram of the Ds Donor Site andPossible Transposition Events

50. 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. 1995 ) • 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