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Transposition and transposable elements

Transposition and transposable elements. Transposable elements. “mobile genetic elements” comprise 45% of human chromosomal DNA “middle repetitive DNA” contribute to spontaneous mutation, genetic rearrangements, horizontal transfer of genetic material

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Transposition and transposable elements

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  1. Transposition and transposable elements

  2. Transposable elements • “mobile genetic elements” • comprise 45% of human chromosomal DNA “middle repetitive DNA” • contribute to spontaneous mutation, genetic rearrangements, horizontal transfer of genetic material • aid speciation and genomic change (in bacteria transposons are often associated with antibiotic resistance genes) • cells must depress transposition to insure genetic stability

  3. Types of transposable elements • DNA vs. RNA • viral vs. nonviral • replicative mechanism vs. excision mechanism

  4. transposon

  5. insertion mutation transposon

  6. Discovery of transposons • Barbara McClintock 1950’s Ac Ds system in maize influencing kernel colorunstable elementschanging map positionpromote chromosomal breaks • Rediscovery of bacterial insertion sequencessource of polar mutationsdiscrete change in physical length of DNAinverted repeat ends: form “lollipops” in EM after denaturation/reannealing

  7. Composite bacterial transposons • repeated ends, usually inverted, sometimes direct • repeated ends themselves are IS elements and can independently transpose • ends mobilize all intervening DNA • often antibiotic resistance genes (examples Tn3 (ampicillin), Tn5 (kanamycin), Tn10 (tetracycline) • often reside on plasmids

  8. Basic minimal insertion sequence structure tnp ORF ends: genetically required, in cis tnp (transposase): genetically required, trans-acting

  9. transposase tnpA “repressor” tnpR Tn3 resolvase ampcillin-resistance bla 38 bp inverted repeat ends 120 bp “IRS” or res internal resolution site Structure of Tn3 4957 bp 3 trans-acting genes: 2 cis-acting sites:

  10. direct repeat of Tn cointegrate tnpR and res mutations cause accumulation of “co-integrate structure”

  11. 2 types of DNA tranposons • excisive mechanismexamples: Tn5, Tn10, P elements • replicative mechanismexamples: Tn3, bacteriophage Mu

  12. Replicative transposons • orignal cut of transposon is only nick and only one strand is initially ligated • element replicates through itself • produces as intermediate a “co-integrate” structure • co-integrate is resolved by resolvase (as TnpR of Tn3) and at specific site (as res of Tn3)

  13. Excisive transposons • cut-and-paste mechanism • cut themselves out of original site, producing double strand break • cut target site and ligate to element ends, thereby inserting at new site • original site break repaired usually with sister chromosome, restoring transposon at original site sometimes end healed without transposon, can also be associated with deletion at excision site

  14. Staggered cleavage of target GAC CTG Ligation of transposon DNA GAC GAC CTG CTG Repair replication generates short direct repeats Source of target site duplication “TSD” GAC CTG

  15. “degenerate” transposons • many naturally occurring transposable elements have suffered mutation and are no longer active • some of these may have cis-acting end mutations and cannot be mobilized • others may have intact ends but no transposase: these can be mobilized by a element that is tnp+ (“autonomous” element) • Ac Ds system is an example of latter: Ac (activator) can mobilize Ds (dissociator) • MITEs (minature inverted repeat transposable elements) are nonautonomous DNA elements • SINEs are retrotransposon version (LINEs)

  16. Comparison of transposition reactions Direct transesterification reactions DDE motif transposase (integrase)

  17. Comparison of tranposase structural organization

  18. Mechanism of transposases and retroviral integrases

  19. Classification of retroelements • Have obligate RNA intermediate, use reverse transcriptase (RT, RNA-dependent DNA polymerase) • LTR-retroelements: long terminal repeatsTy1/copia, Ty3/gypsy, retroviruses • Non-LTR-retroelements “retroposons”LINES

  20. Characteristics of LTR retroelements • Long terminal repeats: required for replication cycle • Genes: gag, pol, (viruses also have env) • Pol is polyprotein which gives rise to RT (reverse transcriptase), IN (integrase) RH (RNase H), PR (protease) • Forms VLPs virus-like particles • Integrase is functionally and structurally similar to transposase of DNA transposons, DDE motif • Integration gives characteristic TSD

  21. LTR element replication tRNA primer Multiple template “jumps”

  22. Characteristics of non-LTR retroelements • 2 ORFs, orf1, orf2 • Variable TSD • ORF2 gives rise to EN, endonuclease, (similar to APE) and RT • Uses target primed reverse transcription TPRT • Can transduce 3’ downstream non-element segments

  23. Target-primed reverse transcription Explains: Insertions are often 5’ truncated Transduction of 3’ markers

  24. Human L1 (LINE-1) retroelement • 15% of human DNA • 520,000 copies, only 3-5,000 are full-length • Associated with human disease loci • Transpose specifically in germ line

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