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)

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 ofthe 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 ofthe 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 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

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 RecombinationGene Knockout in FungiHamer et al. Proc Natl Acad Sci U S A. 2001 24;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 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

40. 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)

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)Systemdiscovered by B. McClintock (Noble Prize Winner in 1983)

48. Ac/Ds System

49. Ac/Ds System

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