Transposable Genetic Elements

1. Transposable Genetic Elements MBIOS 520/420 September 22, 2005

2. MBios 420/520 Announcements • PowerPoint lectures are now available for download at the • web site http://mbios420.tripod.com/mbios420.htm • No longer holding office hours; please e-mail me at • marter@wsu.edu for an appointment • Thoughts on a review session? • Excellent source for questions and review: Go to http://www.ncbi.nlm.gov Search in BOOKS. Enter keywords. Hit GO.

3. Transposon Introduction • Transposable elements are stretches of DNA that can move • to new locations in a genome • These elements can contain genes or be non-coding • Large portions of higher eukaryotes’ genomes are composed • of either inert or active transposons (often as repetitive DNA) • Transposons are thus important evolutionarily • Transposons can also be used to isolate genes or introduce • foreign genes into cells

4. Bacterial Transposon Discovery • First transposons characterized, these are the simplest • Detected because experimental lac- strains kept reverting • back to wild type (ie, colonies kept turning blue) • lac- mutants were due to transposons which then moved back • out of the gene Agar w/X-Gal Agar w/X-Gal transposon Transposition event lac gene lac gene lac gene

5. Bacterial Transposons • Can occur in bacterial genome or in plasmid, and can move between these two • Consist of two major types: • Insertion Sequences (IS) small, <2500 bp • Composite Transposons large, flanked by • two IS elements

6. Insertion Sequences • Consists of a pair of inverted terminal repeats at each end • (cannot be mutated without loss of transposition activity) • Between this is a stretch of DNA, often containing the gene • for transposase – the enzyme that catalyzes transposition • Flanking the terminal repeats are a pair of direct repeats that • result from the transposition process

7. Insertion Sequence Transposition Transposase moves the element by creating a staggered cut at either end in a random spot of the genome The IS element then moves then inserts into this region DNA polymerase fills in the resulting single- stranded areas The result is termed a target site duplication

8. Composite Transposons • Denoted Tn • Created when two IS elements insert • near each other • The elements can be either in inverse or • direct orientation to each other • These two then move together and • transpose the sequence between them • (often carrying genes) • Movement of these large elements is • how bacteria become antibiotic • resistance (often using viral • intermediates)

9. Composition Transposon Transposition • Involves both IS elements • Two types: • Replicative Transposon transposon is copied & moved • (ie, a copy remains in place) • Non-Replicative Tranposon the whole element is moved • (aka “cut & paste”) (no copy remains) • Similar to conserved & semi-conservative DNA replication

10. Tn3 Transposition • A combination of replication & recombination

11. Tn10 Transposition How can we determine if a transposon uses replicative or “cut and paste” transposition?

12. Gene Tagging with Transposons • We can use transposons to tag and isolate genes • Ex: let’s say we want to isolate a blue flower color gene STEP 1 transposon A. Transform plant with a vector containing a transposon. B. Grow progeny from that plant and pick out the mutant phenotype (in this case, an uncolored flower). M2 (Thousands of plants needed, depending on genome size) C. This plant should have the transposon inserted somewhere in the gene. transposon Blue color gene

13. Gene Tagging with Transposons STEP 2 A. Isolate genomic DNA from the mutant plant. B. Make genomic DNA library from this sample (ex: using BAC vectors). BAC clones (many thousands of these) C. Pool clones of the library into 96-well plate. THE MATH Ex: Rice Genome size = 400 million bases BAC insert size = 200,000 bases # clones needed for 1X = 2,000 # clones needed for 6X = 12,000 # of clones per well = 125

14. Gene Tagging with Transposons PCR w/ transposon primers 1 2 3 4 5 STEP 3 1 2 3 4 5 A. Find the well containing the BAC clone with the transposon (use PCR). B. Grow the cells from this well on a plate. P32-labeled transposon probe C. Hybridize with transposon probe to locate exact colony that has the clone. D. Sequence this clone. You’ve found the gene!

15. Gene Tagging with Transposons & Inverse PCR • There is a faster way of identifying the gene without having to • build an entire library, by using a technique called inverse PCR STEP 2b A. Follow STEP 1 like before. Isolate mutant & extract its DNA. EcoRI B. Cut DNA with restriction enzymes, then re-ligate to form circular segments. DNA ligase (high volume reaction) C. Use the transposon-based primers & do PCR. This amplifies the flanking gene regions. Sequence it. SEQUENCE

16. Gene Tagging with Transposons - Troubleshooting SEQUENCE (GENBANK) • What if we get many transposons inserting into our mutant • genome? How do we tell which one is in our color gene? OPTION 1 Do inverse PCR as in STEP 2B. BLAST sequence & search for similarity with other known pigment genes.

17. Gene Tagging with Transposons - Troubleshooting P X F1 F2 A B C D E F λ HindIII Marker 23.0 kb 9.4 kb 6.5 kb 4.3 kb 2.3 kb 2.0 kb 0.5 kb OPTION 2 Cross mutant plant back to wild-type plant. Produce an F2 (or more) generation. Do Southern blot with tranposon probe. Find marker that segregates with mutation. Many plants needed. Make a library out of a plant with only this marker (Plant D in our example).

18. Gene Tagging with Transposons - Troubleshooting A B C D λ HindIII Marker F2 Mutant Plant A B C D 23.0 kb 9.4 kb 6.5 kb 4.3 kb 2.3 kb 2.0 kb 0.5 kb OPTION 3 Pick out all BAC clones with transposons via PCR (STEP 3). Do RFLP ofF2 mutant plant using transposon as a probe. Cut BACs with same restriction enzyme as in your RFLP. Hybe with tranposon probe. Transposon probe should bind at same MW in mutant and BAC digest.

19. Eukaryotic Transposons • Similar in structure to bacterial transposons • Most are thought to be derived from viral genomes that have • integrated into a host cell genome • Some eukaryotic transposons move via an RNA intermediate • Some transpositions are utilized for programmed genome • rearrangements • Movement of transposons in genomes can inactive or activate • genes, and can cause cancer • The movement and buildup of transposable sequences has • had an effect on the evolution of eukaryotice genomes

20. Maize Transposons Transposase Gene Transposase Missing • Two best characterized transposons are Ac (Activator) and Ds • (Dissociation) elements (both are “cut & paste” types) • Can occur in many copies of the cell, but must work together • Ac is ~4.6 kb, with same basic • structure as insertion elements • Ds is similar (identical inverted • terminal repeats) but has • deletions of various sizes in it • Because of the deletions, Ds • does not have transposase & • cannot transpose itself (Ac • needed)

21. Discovery of Transposons CI Present CI Absent • Barbara McClintock, • maize geneticist • Observed mosaic corn • kernels, despite presence of • CI, a dominant colorless allele • Concluded that chromosome • was breaking, causes CI loss • Only occurred when another • segregating factor, Ac, was • present

22. Using Ac & Ds Elements for Mutagenesis Ac+/+ Ds-/- Ac-/- Ds+/+ P X Ac+/- Ds+/- F1 F2 Ac-/- Ac+/? Ds+ X BC1F1 Ac-/- Ds+ Stable Mutant BC1F2 • When we do transposon • mutagenesis, how can we • control when transposition • stops & starts? • Ac can be introduced via a • vector • But how do we know that • Ac won’t transpose into our • gene instead of Ds? Mutate the inverted terminal repeats of Ac, then it can’t transpose!

23. Drosophila Transposons • Known as P elements, similar in structure to Ac & Ds • P elements can be incomplete (no transposase) or complete • (functional transposase) – analogous to Ac/Ds • P elements are only active in • germ line cells, because a stop • codon exists in transposase • In germ cells, alternative splicing • removes exon 2 to remove the • codon • Demonstrated by engineering a P • element without intron 3

24. Retro-Transpososons • These transpose via an RNA intermediate • Transposon is transcribed into RNA, then reverse • transcriptase creates “cDNA” that inserts into genome • Two categories exist, based on their origin & structure: Retroviruslike Elements Possess Long Terminal Repeats Have viral genes gag & pol Derived from retroviruses Retroposons Non-LTR retrotransposons Have poly A:T tract Derived from reverse transcribed mRNA

25. Retroviruslike Elements • Ty1 of yeast is best studied • Long terminal repeats called δ • regions (not inverted) flank a • coding region • Coding region has TyA & TyB; • these genes are gag & pol derived • ~35 copies per yeast cell; • sometimes solo δ regions are • found (formed by recombination • between δ) • Form target site duplications

26. Ty1 Transposition • RNA is synthesized by normal • RNA pol II transcription • δ elements act as strong • promoters (can activate genes) • TyB gene has reverse • transcripase activity and • produces dsDNA from the RNA • DNA integrates into the genome • (δ element is replicated) • copia and gypsy are Drosophila • retrotransposons very similar to • Ty1 (gypsy even has viral env • gene)

27. Proof of Ty1 Transposition • How can we prove Ty1 transposes as an RNA molecule? • Constructed Ty1 element with a galactose-inducible promoter • and an intron • Used galactose to stimulate transcription, then found that all • the new copies transposed had the intron spliced out

28. Retroposons • F, G and I elements in Drosophila; LINEs in humans • Also called non-LTR retrotransposons because they lack • inverted or direct repeats at their ends (do have target site • repeats) • Retroposons all have a poly-A region at the end, evidence that • these are reverse transcribed mRNAs that re-inserted in the • genome • These function by reverse transcription, followed by insertion • LINE-1 or L1 = only known active human transposon (make up • 5% of human genome) & can cause mutations (ex: hemophilia)

29. Transposition & Chromosome Rearrangement Deletions & Duplication Created by Transposition: • Can create duplications, • deletions, inversions, • translocations • Means of creating • pseudogenes – no • selection pressure (can • gain novel function)

30. Transposition & Chromosome Rearrangement Deletions & Inversions Created by Transposition:

31. Transposition & Cancer • Antibody genes have powerful enhancers and use • recombination to produce diverse sets of antibodies • c-myc can recombine with this region & cause cancer

32. Enhancer Trapping Or do inverse PCR • Technique use transposons • to identify tissue-specific • enhancers • Add lacZ gene between • the inverted terminal repeats • of a P element • Transform Drosophila • with P element • Dissect Drosophila & grind • tissue in X-Gal • Blue color change shows • that P element inserted near • a tissue specific enhancer

33. Transposons & Genome Evolution • All active transposons have the potential to cause mutations • These can be deleterious or potentially beneficial • Duplication of genes via chromosome rearrangements can • produce pseudogenes which can eventually gain new function • Which came first  Retroviruses or Retrotransposons?