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Gene Technology

Mobile DNA Sequences in the Genome. Gene Technology. Lecture 8 – Chapter 7. Barbara McClintock (nobel prize winner 1983) Found in the late 1940s -> genetic elements in maize can direct their own movement within the genome. Mobile DNA Sequences in the Genome. Gene Technology.

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Gene Technology

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  1. Mobile DNA Sequences in the Genome Gene Technology Lecture 8 – Chapter 7 Barbara McClintock (nobel prize winner 1983) Found in the late 1940s -> genetic elements in maize can direct their own movement within the genome

  2. Mobile DNA Sequences in the Genome Gene Technology Mobile Elements: -> can be found in all organisms -> 50% of human genome -> interfere with gene expression -> generating mutations (-> Evolution) -> reorganize genomic structure Lecture 7 – Chapter 7

  3. Mutation can be caused by insertions: Mutation in the gal operon was moved to λ phage Gene Technology Insertion

  4. Mobile elements -> Intergration into genome Gene Technology

  5. Principle of mobile elements -> Intergration into genome Gene Technology Transposase-> Enzyme coded on mobile elements -> resposible for excition and integration

  6. Insertion Element (IS): -> most simple mobile elements -> found mainly in bacteria Gene Technology IS element is cut out and leaves behind flanking repeats (non replicative) -> orginal target sequence are increased after jumb

  7. Insertion Element (IS): Gene Technology

  8. Transposons (TN): -> More complex mobile elements -> found in all organisms Transposons: Transposons carry frequently antibiotic resistence genes Transposons can jump from genome to phage or conjugative plasmid -> gene transfere to other bacteria (problem with antibiotica resistent bacteria) Gene Technology

  9. Transposons Gene Technology Composite Transposon: -> Insertion elements on both end of the transposon (one active transposase – one inactive transposase) Simple Transposons: -> Inverted repeats on both ends -> Transposase and Resolvase -> responsible for transfer

  10. Transposons Simple Transposon (Tn3): -> Replicative transposition -> after the jump both have a copy of the transposon Composite Transposons (Tn10): ->non replicative (conservative) -> like tranposition of IS elements Gene Technology

  11. Transposons Gene Technology

  12. Transposons Simple Transposon (Tn3): -> Replicative transposition -> after the jump both have a copy of the transposon Transposase: responsible for excition and transfer Resolvase: responsible for resolution -> replicative transfer Gene Technology

  13. Transposons Gene Technology An R plasmid may contain several transposons carrying (resistance genes)

  14. Transposons Composite transposons: -> non replicative transfer Ac element (first discovered in maize) -> Activator (avtive transposon) Ds element (first discovered in maize) -> Dissociation -> passive transposon-> needs Ac to move Gene Technology

  15. Transposons Composite transposons: -> non replicative transfer Ac element (first discovered in maize) -> Activator (avtive transposon) Ds element (first discovered in maize) -> Dissociation -> passive transposon-> needs Ac to move Gene Technology Deletions in Ac element -> gives Ds element -> inactive transposase -> needs Ac to move

  16. Mechanism of Ac or Ds tranfer Gene Technology

  17. Transposons Transposition has influence on phenotype of maize First recognized by Barbara McKintock (first transposon found) Gene Technology Movement of Transposon during development of fruit -> pattern in pigmentation

  18. Transposons Gene Technology

  19. Transposons Transposons are activated by hemimethylated DNA -> right after replication when second strand is not yet methylated When non replicative transposon (Tn10) jumps it leaves a double strand break behind -> repaired by using other stand (with transposon in) as template (repair pathway) -> in the end both strand have the transposon in 2 times Not only both cells after replication have the transposon -> also number of transposons increased!!!! Gene Technology

  20. Retrotransposons are similar to retroviruses RNA intermediate + use of reverse transcriptase Gene Technology

  21. Retrotransposons are similar to retroviruses RNA intermediate + use of reverse transcriptase Gene Technology

  22. Transposons versus Retrotransposon Gene Technology

  23. Retrotransposon • There are 2 groups: • LTR (long terminal repeats) retrotransposons (viral like) • Non-LTR retrotransposons (polyA) Gene Technology Carry strong promoters If transposon inserted 5’ to silenced gene -> activates gene expression of these genes Retrotransposons can cause cancer!! Promoter necessary for transposition

  24. Retrotransposon Protein coded for : Integrase + Reverse transcriptase Gene Technology Carry strong promoters for RNA ploymerase If transposon inserted 5’ to silenced gene -> activates gene expression of these genes Retrotransposons can cause cancer!! Retroviruses can also cause cancer!! ORF1: RNA binding protein ORF2: Endonuclease + reverse transcriptase

  25. Retrotransposon Demonstration that retrotransposon transfers through RNA intermediate Ty is yeast LTR retrotransposon Gene Technology

  26. Non-LTR Retrotransposon ORF1: RNA binding protein ORF2: Endonuclease + reverse transcriptase Gene Technology Promoter necessary for transposition mRNA transcript with poly A

  27. Non-LTR Retrotransposon Transposition -> target-primed reverse transcription Reverse transcription commonly stops before element has been fully transcribed -> a lot of truncated L1 elements Alu elements: 300bp repetitive sequences (11% of human genome) -> belong to SINEs (do not encode their own reverse transcriptase -> nonautonomous transposons SINEs are probably trancated LINEs (share same 3’ sequence) Alu elements are activated by LINEs Gene Technology

  28. Gene Technology

  29. Types of transposable elements in the human genome Gene Technology

  30. Human genes contain many transposable elements Gene Technology Important for survival -> Exon/Intron concept of a gene

  31. Transposons are mainly responsible for differences in genome size Gene Technology

  32. Gene Technology

  33. Transposons can cause Mutations Transposable elements at work in snapdragon Result of transposition or excision -> not always clean excited -> has an effect on enhancer -> different expression level Gene Technology

  34. Transposons can cause Mutations Transposons and retrotransposons can induce mutations by inserting near or within genes. -> transposon-induced mutations are relatively stable, because the sequence at the insertion site is retained as they transpose via the replication mechanism. Gene Technology Most mutations caused by transposons are deleterious -> L1 insertion into hemophilia A Retrotransposons can be responsible for genetic disease (34 diseases identified) -> hemophilia, muscular dystrophy, … Development of genetic disease can happen by transposon jumb in the embryo!!!

  35. Transposons generate diversity for evolution Gene Technology

  36. Transposons generate diversity for evolution Can affect genes (delection, insertion, exchange), regulatory elements Gene Technology

  37. Transposons generate diversity for evolution Can affect genes (delection, insertion, exchange), regulatory elements Gene Technology Mutant receptors -> cannot remove cholesterol from blood

  38. Transposons can be silenced Organisms with high transposition (Drosphila) can silence transposition -> P element encodes not only transposase but also an inhibitor to transpostion (Protein alters slicing of transposase -> inactive transposase) Gene Technology

  39. Transposons can be silenced Gene Technology If female has P element -> oocytes (eggs) with inhibitor produced -> normal flies If female has no P element -> oocytes have burst of transposition in embryo -> steril offspring

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