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LECTURE 18: TRANSPOSABLE ELEMENTS

LECTURE 18: TRANSPOSABLE ELEMENTS. chapter 13 exam 2 & grades general ideas discovery in maise prokaryotes eukaryotes dynamic & plentiful host regulation. The Biology Graduate Student Association Presents.

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LECTURE 18: TRANSPOSABLE ELEMENTS

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  1. LECTURE 18: TRANSPOSABLE ELEMENTS • chapter 13 • exam 2 & grades • general ideas • discovery in maise • prokaryotes • eukaryotes • dynamic & plentiful • host regulation

  2. The Biology Graduate Student Association Presents A forum to present current and prospective research projects in the field of Biology Barrick Museum Auditorium Saturday, November 18th 8:00 am to 2:30 pm

  3. LECTURE 21 TRANSPOSABLE ELEMENTSQUIZ #5 • you need a piece of paper and a pen or pencil... • write your name and student number at the top... • give brief answers for the questions below... Q1: Transposable elements were first discovered in _________. Q2: Name the 2 classes of eukaryotic transposons and describe some of their defining features.

  4. TRANSPOSABLE ELEMENT QUESTIONS • why were they 1st discovered in corn but 1st isolated from E. coli? • how do they confer antibiotic resistance in bacteria? • why the classification as DNA orRNA transposons? • what do autonomous and non-autonomous mean? • what are the implications for the human genome with  50% of it derived from transposable elements

  5. GENERAL IDEAS • Barbara McClintock 1920s 1980s (nobel in 1984) • mostly ignored for decades •  50% of human genome • 2 types class 1. retrotransposons • transcribe class 2. DNA elements • excise

  6. DISCOVERY IN MAISE • Barbara McClintock 1920s 1980s (Nobel in 1983) • “Indian” corn, 10 chromosome pairs • Dissociation (Ds) factor on # 9 commonly broken • Activator (Ac) unlinked factor required for breaks • could not map to constant place • lots of bizarre kernel phenotypes, not ~ parents

  7. DISCOVERY IN MAISE • mosaicism through the activity of Ds movement • during mitosis, results in patchy tissue • chromosome breakage & loss • all linked genes affected

  8. DISCOVERY IN MAISE • mosaicism through the activity of Ds movement • during mitosis, results in patchy tissue • unstable insertions & excission • single gene affected only, e.g.C gene

  9. DISCOVERY IN MAISE • mosaicism through the activity of Ds movement • during mitosis, results in patchy tissue • size of mutant sector ~ time of event

  10. DISCOVERY IN MAISE • autonomous & nonautonomous elements • C gene, allele called c-mutable(Ds) or c-m(Ds) • Ds stable without Ac • Ds excision is dependent on Ac • Ds is nonautonomous • C gene, allele called c-mutable(Ac) or c-m(Ac) • Ac always unstable • Ac is autonomous • Ac can transform Ds • Ds = Ac mutant

  11. DISCOVERY IN MAISE • autonomous & nonautonomous elements • Ds & Ac are members of a transposable element family • many other families discovered in maize • autonomous elements encode information necessary for the transposition of themselves and nonautonomous members of their family

  12. PROKARYOTES • molecular biology of transposable elements first characterized in bacteria • insertion sequence (IS) elements • transposable • can block gene & operon function (polar) • e.g., gal operon in E. coli

  13. PROKARYOTES • are the gal mutants all the same?... NO • several different IS sequences • inserted in different places • all encode transposase enzyme • multiple copies,  can recombine F factor

  14. PROKARYOTES • 2 types of bacterial transposons • composite: genes (e.g., drug resistance) sandwiched between inverted IS sequences... • in this case, these are called inverted repeat (IR) sequences

  15. PROKARYOTES • 2 types of bacterial transposons • simple: genes sandwiched between IR sequences • genes include transposase • IR sequences are short (<50 bp) & do not encode transposase

  16. PROKARYOTES • transposons can tranpose (jump) to & from plasmids & chromosomes • implications for drug resistance

  17. PROKARYOTES • basic mechanism of tranposition • transposase makes staggered cuts in host DNA • element inserts • host DNA repair fills in gaps • in this example, it generates 5-bp direct repeats on either side • called target-site duplications

  18. PROKARYOTES • 2 types (at least) of transposition mechanisms • replicative: copy remains in original site • conservative (nonreplicative): excision only

  19. PROKARYOTES • 2 types (at least) of transposition mechanisms • replicative: copy remains in original site • recombination event

  20. EUKARYOTES • 2 types of eukaryotic transposons class 1. retrotransposons • transcription mechanism class 2. DNA transposons • excision mechanism

  21. EUKARYOTES class 1. retrotransposons • resemble single stranded RNA retroviruses • copied into DNA using reverse transcriptase • inserts into host • transcribes new viral genome & proteins  new viral particles • called provirus when integrated

  22. EUKARYOTES class 1. retrotransposons • resemble single stranded RNA retroviruses • similar structure & gene content • flanked by long terminal repeat sequences (LTRs) 100s of bp long • these are also called LTR-retrotransposons

  23. EUKARYOTES class 1. retrotransposons • gag: maturation of RNA genome • pol: reverse transcriptase • env: protein coat (viral gene only)

  24. EUKARYOTES class 1. retrotransposons • Ty elements in yeast • copia-like elements in Drosophila • 10-100 positions in genome • cause known mutations, e.g., wa w+wa w1118

  25. EUKARYOTES class 2. DNA transposons • mechanisms similar to those in bacteria • DrosophilaP-elements 1st characterized • discovered ~ hybrid dysgenesis

  26. EUKARYOTES class 2. DNA transposons • mechanisms similar to those in bacteria • DrosophilaP-elements first characterized • discovered ~ hybrid dysgenesis • P-strains have 30 - 50 P-element copies / genome • 2.9 kb wild type element, 31 bp inverted repeats • defective elements are smaller • tranposase gene has 3 introns + 4 exons

  27. EUKARYOTES class 2. DNA transposons • hybrid dysgenesis mechanism in Drosophila

  28. EUKARYOTES class 2. DNA transposons • action of Ac element in maise

  29. EUKARYOTES • DNA transposons, gene discovery & manipulation • controlled use of engineered P-elements • 2 element system: • 2-3: • transposase source • disrupted terminal IR sequences • stable (immobilized) • bullet: • deleted transposase gene • inserted genes of interest (e.g. markers) • mobilized only in combination with #1

  30. EUKARYOTES • DNA transposons, gene discovery & manipulation • controlled use of engineered P-elements • genes of interest inserted in bullet • gene transfer...  • re-mobilization

  31. EUKARYOTES • DNA transposons, gene discovery & manipulation • controlled use of engineered P-elements • insertional mutagenesis • provide transposase for 1 generation • cross away & screen for new mutants • use P-element sequence to probe for gene = transposon tagging • enhancer trap mutagenesis • finds functional regulatory sequences • GAL4 system (binary, 2 bullets) • tool for gene manipulation

  32. /   w– E X GAL4 w+ x GFP w+ / w– P-ELEMENTS & YEAST GAL4 SYSTEM

  33. GAL4 w+ GFP w+ green fluorescent protein P-ELEMENTS & YEAST GAL4 SYSTEM / w– E X / w–

  34. P-ELEMENTS & YEAST GAL4 SYSTEM MUSHROOM BODY KENYAN CELL CYTOPLASMIC SIGNAL NUCLEAR SIGNAL

  35. DYNAMIC & PLENTIFUL • DNA content of organism  C-value • lack of correlation with biological complexity C-value paradox • DNA repeat sequences make up large fraction of eukaryotic genomes • genome size correlates with amount of DNA derived from transposable elements • e.g., ~ half of the human genome is derived from transposable elements

  36. DYNAMIC & PLENTIFUL • human genome • long interspersed nuclear elements (LINES) • autonomous, retrotranspose, no LTRs • short interspersed nuclear elements (SINES) • nonautonomous, ~ lines w/o rev. transcriptase • Alu element ~ 10% of genome

  37. DYNAMIC & PLENTIFUL • human genome • ~ 20  as much DNA derive from transposable elements as protein-encoding DNA • intron insertions remain only  spiced out • presumably initially also in exons  mutations & negative selection • typical pattern in humans...

  38. DYNAMIC & PLENTIFUL • human genome • class 1 transposons (LINES, SINES) cause some hereditary diseases in humans, e.g., • hemophilia A • neurofibromatosis • breast cancer • class 2 transposons (DNA) • low mutation rate (0.2 % or 1 in 500 known)

  39. DYNAMIC & PLENTIFUL • plants (e.g. grasses) • synteny: similar gene content & organization • vastly different genome sizes due to transposons • safe havens: strategy of insertion in other transposons, minimize negative effect on host transposons genes

  40. DYNAMIC & PLENTIFUL • yeast • small genome, 70% exons • Ty LTR-retrotransposons • targeted insertions to benign sites • encoded integration enzyme

  41. DYNAMIC & PLENTIFUL • Drosophila • telomeres are transposable elements! • HeT-A & TART non-LTR retrotransposons (LINES) • telomerase is a reverse transcriptase • RNA template for telomere DNA synthesis

  42. HOST REGULATION • Ac activity reversible • lost of activity reappeared in later generations • epimutations: changes in chromatin structure

  43. HOST REGULATION • transgene silencing • cosuppression: transformed gene & endogenous homologous genes both silenced • unknown defense mechanism?

  44. SPEND SOME TIME WITH... • key questions revisited (p.446-447) • summary (p.447) • terminology (p.447-8) • unsolved problems (p.449)... • 2, 3, 5, 7, 11

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