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  • Left image are sketches of McClintock’s observation of chromosomes in Maize.
  • The majority of transposition events are ancient, but in Sue Wessler’s story, we se tranpsons in action.
  • transposons complete ppt

    1. 1. Transposable elements 1
    2. 2.  The Nobel Prize in Physiology or Medicine 1983 was awarded to Barbara McClintock "for her discovery of mobile genetic elements". Barbara McClintock
    3. 3. The Dynamic Genome Transposons 3
    4. 4. Transposons and Insertional Mutations Transposons: Mobile Genetic Elements Transposon Transposon TransgenesisBarbara McClintock Transposon Insertional Transposon Mutagenesischromosome 基 Gene 因 Mutant Gene Tagged 4
    5. 5. Advantages of Insertional Mutations  can produce easily tractable mutations  can produce large number of mutants at low cost and high speed 5
    6. 6. What are Transposons?Transposable element (transposon): a sequence of DNA that is comfrom place to place within a genome Transposition of DNA on chromosome 9 of maize explains mottl 6 Some definitions and figures from Lisch 2009: Annu. Rev. Plant Biol. 2009.60:43-66.
    7. 7. What are Transposons? Transposable element (transposon): a sequence of DNA that is com from place to place within a genome(1) At the beginning of kernel development, the Ds transposon isinserted into the colored (C) gene, resulting in colorless tissue. (2) Dstransposition early in kernel development restores the C gene, givingrise to a large colored sector. (3) Transposition later in kerneldevelopment results in smaller sectors. 7 Learn more at:
    8. 8. What are Transposons?Transposable element (transposon): a sequence of DNA that is comfrom place to place within a genome “Cut & Paste” “Copy & Paste” 8
    9. 9. What are Transposons?• Plant genomes contain multiple transposon families.• Each contains autonomous and non-autonomous elements.• Class I transposons do not move, but are being copied.• Class II transposons move, but can undergo copying, too (if transposing during DNA replication) Autonomous element Gene( s) Nonautonomous elements 9
    10. 10. What are Transposons? Transposons make up the major content of eukaryotic genomes• ~50% of the genomes of human, chimp, mouse, ape• ~75% of the maize genome• ~85% of the barley genome• ~98% of the iris genome Iris brevicaulis Iris fulva 10
    11. 11. What are Transposons?Variation in cereal genomes - transposons & genome duplications Sorghum 700 Maize 2,500 Mb Rice 450 Mb MbBarley 5,000 Mb Wheat 20,000 Mb Oats ~20,000 Mb 11
    12. 12. Transposons in Action 12
    13. 13. How do organisms live with TEs?• Most TEs are broken (cannot tranpose; “fossils”).• Active TEs evolved to insert into “safe-havens.”• Host regulates TE movement.• TEs can provide advantages. 13
    14. 14. Ping/mPingmPing: MITEs are being amplified to high copy numbersMITE (Multi-insertional TE)Deletion-derivative of PingRequires Ping transposase to jump 14
    15. 15. mPing copy number in O.japonica OVER 1000 mPing copies mPing Japonica strains Over 1000 copies of mPing in 4 related strains…. 15 Naito et al PNAS (2006)) Takatoshi Tanisaka lab (Kyoto
    16. 16. Genomic distribution of mPing insertions • predominantly in genic regions in euchromatin • even inserts in heterochromatin are in genes • where does mPing insert in and around genes? 16
    17. 17. Genic distribution of mPing insertions 12 shared (n=926) 10 unshared 8 (n=736) expect. (%) 6 4 2 0 5 TR U exon i ntron 3 TR U UTR Exon UTR 17 mPing insertions rare in coding-exons
    18. 18. TEs can alter gene expression Os02g0135500 (-41)2.5 NB EG4 (mPing+) 2 A123 (mPing+) A1571.5 10.5 0 control cold salt dry 18 mPing found to confer cold and salt inducibility
    19. 19. TEs can alter gene expression Can this have phenotypic consequences? Nipponbare EG4 19 EG4 is salt tolerant
    20. 20. Rapid mPing amplification (burst)• Massive amplification largely benign• Subtle impact on the expression of many genes• Produces stress-inducible networks (cold, salt, others?)• Generates dominant alleles Naito et al, Nature, 2009 20
    21. 21. TEs as tools of evolutionary change• TEs usually inactive.• “Stress” conditions may activate TEs.• Active TEs increase mutation frequency.• Most mutations caused by TEs neutral or harmful.• A rare TE-induced mutation (or rearrangement) may be adaptive. Transposable elements can shake up otherwise conservative genomes and generate new genetic diversity. 21
    22. 22. TEs for student research projects • (relatively) simple • incredibly abundant • evolve rapidly • promote rapid genome evolution • largely ignored (discovery) 22
    23. 23. Transposons Fall into two general classes with respect to how they move. One class encodes proteins that move the DNA element directly to a new position or replicate the DNA. – Found in both prokaryotes and eukaryotes The other class are related to retroviruses in that they encode a reverse transcriptase for making DNA copies of their RNA transcripts, which then integrate at new sites in the genome. – Found only in eukaryotes. 23
    24. 24.  Transposable elements are important because they can insert into sites where there is no sequence homology (nonhomologous recombination) 24
    25. 25. Prokaryotes What are two types of transposons in prokaryotes and how do they differ? (IS and Tn) – What enzyme is required for the transposition of an IS element? – How is a composite transposon different from a noncomposite transposon? – How does the replicative transposition mechanism differ from the conservative mechanism of 25
    26. 26. 26
    27. 27. 27
    28. 28. 28
    29. 29. 29
    30. 30.  EUKARYOTIC TRANSPOSITION What is cytogenetics, and how was it used to find “jumping genes” in eukaryotes? In what ways are eukaryotic transposable elements similar to those found in prokaryotes? What can determine the stability of a newly-inserted transposable element in plants? 30
    31. 31.  What genes do Ty elements in yeast carry, and what are their purposes? In what ways is the yeast Ty element similar to a retrovirus? Why are Ty elements classified as retroposons? 31
    32. 32. 32
    33. 33. Transposable Elements (Transposons) DNA elements capable of moving ("transposing") about the genome Discovered by Barbara McClintock, largely from cytogenetic studies in maize, but since found in most organisms She was studying "variegation" or sectoring in leaves and seeds She liked to call them "controlling elements“ because they affected gene expression in myriad ways 33
    34. 34. 1. Nobel Prize inPhysiology and Medicine)2. Barbara McClintock 1902-1992 34 Corn (maize) varieties
    35. 35. Corn evolution in 7000 yrs of domestication cob of Hopi Blue corn cob of wild teosinte 35
    36. 36. Maize (domesticated corn) kernel structure 36
    37. 37. Mutant Kernel Phenotypes 1. Pigmentation mutants – affect anthocyanin pathway – elements jump in/out of transcription factor genes (C or R) – sectoring phenotype - somatic mutations – whole kernel effected - germ line mutation2. Starch synthesis mutants - stain starch with iodine, see sectoring in endosperm 37
    38. 38. Some maize phenotypes caused by transposableelements excising in somatic tissues. Start with lines that produce kernels defective in starch synthesis (endosperm phenotypes) or anthocyanin synthesis (aleurone and pericarp phenotypes) because of an inserted element, and the 38 element excises during development.
    39. 39. Somatic Excision of Ds from C Wild type Mutant SectoringFig. 23.9 39
    40. 40. Other Characteristics of McClintocks Elements Unstable mutations that revert frequently but often partially, giving new phenotypes. Some elements (e.g., Ds) correlated with chromosome breaks. Elements often move during meiosis and mitosis. Element movement accelerated by genome damage. 40
    41. 41. Molecular Analysis of Transposons Transposons isolated by first cloning a gene that they invaded. A number have been cloned this way, vAia "Transposon trapping“. Some common molecular features: – Exist as multiple copies in the genome – Insertion site of element does not have extensive homology to the transposon – Termini are an inverted repeat – Encode “transposases” that promote movement – A short, direct repeat of genomic DNA often flanks the transposon : “Footprint” 41
    42. 42. Ac and Ds Ds is derived from Ac by internal deletions Ds is not autonomous, requires Ac to move Element termini are an imperfect IR Ac encodes a protein that promotes movement - Transposase Transposase excises element at IR, and also cuts the target 42
    43. 43. Structure of Ac and Ds deletion derivativesDs is not autonomous, requires Ac to move! 43 Fig. 23.10
    44. 44. How duplicationsin the target siteprobably occur. Duplication remains when element excises, thus the Footprint. 44 Fig. 23.2
    45. 45. Mu/MuDR (Mutator) Discovered in maize; differs significantly from Ac and En/Spm families Autonomous and non-autonomous versions; many copies per cell Contain a long TIR (~200 bp) Transpose via a gain/loss (somatic cells) or a replicative (germline cells) mechanism. 45
    46. 46. Structure of MuDR(autonomous Mu)and its promoters.• MuDrA and Bexpressed at highlevels in dividing cellsand pollen, because oftranscriptionalenhancers.• MURA is transposase& has NLS.• MURB needed forinsertion in somaticcells. 46
    47. 47. Retro-TransposonsCan reachhigh numbersin thegenomebecause ofreplicativemovement. 47Fig. 7.34 in Buchanan et al.
    48. 48. Control of Transposons Autoregulation: Some transposases are transcriptional repressors of their own promoter(s)  e.g., TpnA of the Spm element Transcriptional silencing: mechanism not well understood but correlates with methylation of the promoter (also methylation of the IRs) 48
    49. 49. Biological Significance of Transposons They provide a means for genomic change and variation, particularly in response to stress (McClintock’s "stress" hypothesis) (1983 Nobel lecture, Science 226:792) or just "selfish DNA"? No known examples of an element playing a normal role in development. 49
    50. 50. Transposable elements AC and DS in maize – AC encodes transposase, required to excise DS 50
    51. 51. Transposon tagging 51
    52. 52. Transposon tagging utilizescolorimetric expression assays GUS reporter gene (B-glucuronidase), E. coli GFP (green fluorescent protein), jellyfish 52
    53. 53. General Features of Transposable Elements1. Transposable elements are divided into two classes on the basis of their mechanism for movement: a. Some encode proteins that move the DNA directly to a new position or replicate the DNA to produce a new element that integrates elsewhere. This type is found in both prokaryotes and eukaryotes. b. Others are related to retroviruses, and encode reverse transcriptase for making DNA copies of their RNA transcripts, which then integrate at new sites. This type is found only in eukaryotes.2. Transposition is nonhomologous recombination, with insertion into DNA that has no sequence homology with the transposon. a. In prokaryotes, transposition can be into the cell’s chromosome, a plasmid or a phage chromosome. b. In eukaryotes, insertion can be into the same or a different chromosome.3. Transposable elements can cause genetic changes, and have been involved in the evolution of both prokaryotic and eukaryotic genomes. Transposons may: a. Insert into genes. b. Increase or decrease gene expression by insertion into regulatory sequences. c. Produce chromosomal mutations through the mechanics of transposition. Chapter 20 slide 53
    54. 54. Transposable Elements in Prokaryotes1.Prokaryotic examples include: a. Insertion sequence (IS) elements. b.Transposons (Tn). c. Bacteriophage Mu (replicated by transposition) Chapter 20 slide 54
    55. 55. Insertion SequencesAnimation: Insertion Sequences in Prokaryotes1. IS elements are the simplest transposable elements found in prokaryotes, encoding only genes for mobilization and insertion of its DNA. IS elements are commonly found in bacterial chromosomes and plasmids.2. IS elements were first identified in E. coli’s galactose operon, wheresome mutations’ were shown to result from insertion of a DNA sequence now called IS1 (Figure 20.1)3. Prokaryotic IS elements range in size from 768 bp to over 5 kb. Known E. coli IS elements include: a. IS1 is 768 bp long, and present in 4–19 copies on the E. coli chromosome. b. IS2 has 0–12 copies on the chromosome, and 1 copy on the F plasmid. c. IS10 is found in R plasmids.4. The ends of all sequenced IS elements show inverted terminal repeats (IRs) of 9–41 bp (e.g., IS1 has 2355 of nearly identical sequence). Chapter 20 slide bp
    56. 56. Fig. 20.1 The insertion sequence (IS) transposable element, IS1 Chapter 20 slide 56Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
    57. 57. 5. Integration of IS elements may: a. Disrupt coding sequences or regulatory regions. b. Alter expression of nearby genes by the action of IS element promoters. c. Cause deletions and inversions in adjacent DNA. d. Serve as a site for crossing-over between duplicated IS elements.6. When an IS element transposes: a. The original copy stays in place, and a new copy inserts randomly into the chromosome. b. The IS element uses the host cell replication enzymes for precise replication. c. Transposition requires transposase, an enzyme encoded by the IS element. d. Transposase recognizes the IR sequences to initiate transposition. e. IS elements insert into the chromosome without sequence homology (illegitimate recombination) at target sites (Figure 20.2). i. A staggered cut is made in the target site, and the IS element inserted. ii. DNA polymerase and ligase fill the gaps, producing small direct repeats of the target site flanking the IS element (target site duplications). f. Mutational analysis shows that IR sequences are the key Chapter 20 slide 57
    58. 58. Fig. 20.2 Schematic of the integration of an IS element into chromosomal DNA Chapter 20 slide 58Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
    59. 59. Transposons1. Transposons are similar to IS elements, but carry additional genes, and have a more complex structure. There are two types of prokaryotic transposons: a. Composite transposons carry genes (e.g., antibiotic resistance) flanked on both sides by IS elements (IS modules). i. The IS elements are of the same type, and called ISL (left) and ISR (right). ii. ISL and ISR may be in direct or inverted orientation to each other. iii. Tn10 is an example of a composite transposon (Figure 20.3). It is 9.3 kb, and contains: (1) 6.5 kb of central DNA with genes that include tetracycline resistance (a selectable marker). (2) 1.4 kb IS elements (IS10L and IS10R) at each end, in an inverted orientation. iv. Transposition of composite transposons results from the IS elements, which supply transposase and its recognition signals, the IRs. (1) Tn10’s transposition is rare, because transpose is produced at a rate of ,1 molecule/generation. (2) Transposons, like IS elements, produce target site duplications (e.g., a 9- bp duplication for Tn10). (Table 20.1) Chapter 20 slide 59
    60. 60. Fig. 20.3 Structure of the composite transposon Tn10 Chapter 20 slide 60Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
    61. 61. b. Noncomposite transposons also carry genes (e.g., drug resistance) but do not terminate with IS elements. i. Transposition proteins are encoded in the central region. ii. The ends are repeated sequences (but not IS elements). iii. Noncomposite transposons cause target site duplications (like composite transposons). iv. An example is Tn3. (1) Tn3’s length is about 5 kb, with 38-bp inverted terminal repeats. (2) It has three genes in its central region: (a) bla encodes β-lactamase, which breaks down ampiciliin. (b) tnpA encodes transposase, needed for insertion into a new site. (c) tnpB encodes resolvase, involved in recombinational events needed for transposition (not found in all transposons). (3) Tn3 produces 20 slide 61 duplication upon insertion (Figure Chapter a 5-bp 20.5).
    62. 62. Fig. 20.4 Structure of the noncomposite transposon Tn3 Chapter 20 slide 62Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
    63. 63. Fig. 20.5 DNA sequence of a target site of Tn3 Chapter 20 slide 63Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
    64. 64. 2. Models have been generated for transposition: a. Cointegration is an example of the replicative transposition that occurs with Tn3 and its relatives (Figure 20.6). i. Donor DNA containing the Tn fuses with recipient DNA. ii. The Tn is duplicated, with one copy at each donor-recipient DNA junction, producing a cointegrate. iii. The cointegrate is resolved into two products, each with one copy of the Tn. b. Conservative (nonreplicative) transposition is used by Tn10, for example. The Tn is lost from its original position when it transposes.3. Transposons cause the same sorts of mutations caused by IS elements: a. Insertion into a gene disrupts it. b. Gene expression is changed by adjacent Tn promoters. c. Deletions and insertions occur. d. Crossing-over results from duplicated Tn sequences in the genome. Chapter 20 slide 64
    65. 65. Fig. 20.6 Cointegration model for transposition of a transposable element by replicative transposition Chapter 20 slide 65Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
    66. 66. IS Elements and Transposons in Plasmids1. Bacterial plasmids are extrachromosomal DNA capable of self-replication. Some are episomes, able to integrate into the bacterial chromosome. The E. coli F plasmid is an example (Figure 20.7): a. Important genetic elements of the F plasmid are: i. tra genes for conjugal transfer of DNA from donor to recipient. ii. Genes for plasmid replication. iii. 4 IS elements: 2 copies of IS3, 1 of IS2, and 1 of γδ (gammadelta). All have homology with IS elements itt the E. coli chromosome. b. The F factor integrates by homologous recombination between IS elements, mediated by the tra genes.2. R plasmids have medical significance, because they carry genes for resistance to antibiotics, and transfer them between bacteria (Figure 20.7). a. Genetic features of R plasmids include: i. The resistance transfer factor region (RTF), needed for conjugal transfer. It includes a DNA region homologous to an F plasmid region, and genes for plasmid-specific DNA replication. ii. Differing sets of genes, such as those for resistance to antibiotics or heavy metals. The resistance genes are transposons, flanked by IS module-like sequences, and can replicate and insert into the bacterial chromosome. b. R plasmids are clinically significant, because they disseminate drug resistance genes between bacteria. Chapter 20 slide 66
    67. 67. Fig. 20.7 Organizational maps of bacterial plasmids with transposable elements Chapter 20 slide 67Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
    68. 68. Bacteriophage Mu1. Temperate bacteriophage Mu (mutator) can cause mutations when it transposes. Its structure includes: a. A 37 kb linear DNA in the phage particle that has central phage DNA and unequal lengths of host DNA at the ends (Figure 20.8). b. The DNA’s G segment can invert, and is found in both orientations in viral DNA.2. Following infection, Mu integrates into the host chromosome by conservative (non-replicative) transposition. a. Integration produces prophage DNA flanked by 5 bp target site direct repeats. b. Flanking DNA from the previous host is lost during integration. c. The Mu prophage now replicates only when the E. coli chromosome replicates, due to a phage-encocled repressor that prevents most Mu gene expression.3. Mu prophage stays integrated during the lytic cycle, and replication of Mu’s genome is by replicative transposition.4. Mu causes insertions, deletions, inversions and translocations (Figure 20.9). Chapter 20 slide 68
    69. 69. Fig. 20.8 Temperate bacteriophage Mu genome shown in (a) as in phage particles and (b) as integrated into the E. coli chromosome as a prophage Chapter 20 slide 69Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
    70. 70. Fig. 20.9 Production of deletion or inversion by homologous recombination between two Mu genomes or two transposons Chapter 20 slide 70Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
    71. 71. Transposable Elements in Eukaryotes1. Rhoades (1930s) working with sweet corn, observed interactions between two genes: a. A gene for purple seed color, the Al locus. Homozygous mutants (a/a) have colorless seeds. b. A gene on a different chromosome, Dt (dotted) that causes seeds with genotype a/a Dt/-- to have purple dots. i. Dt appears to mutate the a allele back to the Al wild-type in regions of the seed, producing a dotted phenotype. ii. The effect of the Dt allele is dose dependent. (1) One dose gave an average of 7.2 dots per seed. (2) Two doses gave an average of 22.2 dots/seed. (3) Three doses gave an average of 121.9 dots/seed. c. Rhoades interpreted Dt as a mutator gene.2. McClintock (1940s-50s), working with corn (Zea mays) proposed the existence of “controlling elements” that regulate other genes and are mobile in the genome.3. The genes studied by both Rhoades and McClintock have turned out to be transposable elements, and many others have been identified in various eukaryotes. a. Most studied are transposons of yeast, Drosophila, corn and humans. b. Their structure is very similar to that of prokaryotic transposable elements. c. Eukaryotic transposable elements have genes for transposition and integration at a number of sites, as well as a variety of other genes. d. Random insertion results from 20 slide 71 Chapter non-homologous recombination, and means that any chromosomal gene may be regulated by a transposon.
    72. 72. Transposons in PlantsAnimation: Transposable Elements in Plants1. Plant transposons also have IR sequences, and generate short direct target site repeats.2. The result of transposon insertion into a plant chromosome will depend on the properties of the transposon, with possible effects including: a. Activation or repression of adjacent genes by disrupting a cellular promoter, or by action of transposon promoters. b. Chromosome mutations such as duplications, deletions, inversions, translocations or breakage. c. Disruption of genes to produce a null mutation (gene is nonfunctional).3. Several families of transposons have been identified in corn, each with characteristic numbers, types and locations. a. Each family has two forms of transposon. Either can insert into a gene and produce a mutant allele. i. Autonomous elements, which can transpose by themselves. Alleles produced by an autonomous element are mutable alleles, creating mutations that revert when the transposon is excised from the gene. ii. Nonautonomous elements, which lack a transposition gene and rely on the presence of another transposon to supply the missing function. Mutation by these elements is stable (except when an autonomous element from the family is also present). Chapter 20 slide 72
    73. 73. 4. Multiple genes control corn color, and classical genetics indicates that a mutation in any of these genes leads to a colorless kernel. McClintock studied the unstable mutation that produces spots of purple pigment on white kernels (Figure 20.10). a. She concluded that spots do not result from a conventional mutation, but from a controlling element (now Tn). b. A corn plant with genotype c/c will have white kernels, while C/-- will result in purple ones. i. If a reversion of c to C occurs in a cell, that cell will produce purple pigment, and hence a spot. ii. The earlier in development the reversion occurs, the larger the spot. Chapter 20 slide 73
    74. 74. iii. McClintock concluded that the c allele resulted from insertion of a “mobile controlling element” into the C allele. (1) The element is Ds (dissociation), now known to be a nonautonomous transposon. (2) Its transposition is controlled by Ac (activator), an autonomous transposon (Figure 20.11).c. McClintock’s evidence of transposable elements did not fit the prevailing model of a static genome. More recent studies have confirmed and characterized the elements involved. i. The Ac-Ds system involves an autonomous element (Ac) whose insertions are unstable, and a nonautonomous element (Ds) whose insertions are stable if only Ds is present. ii. McClintock (1950s) showed that some Ds elements derive from Ac elements. Chapter 20 slide 74
    75. 75. Fig. 20.11 Kernel color in corn and transposon effects Chapter 20 slide 75Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
    76. 76. iii. Ac is 4,563 bp, with 1 1-bp imperfect terminal IRs and 1 transcription unit producing a 3.5 kb mRNA encoding an 807 amino acid transposase. Insertion generates an 8-bp target site duplication (Figure 20.12).iv. Ac activates Ds to transpose or break the chromosome where it is inserted.v. Ds elements vary in length and sequence, but all have the same terminal IRs as Ac, and many are deleted or rearranged versions of Unique to corn transposons, timing and frequency of transposition and gene rearrangements are developmentally regulated.vii. Ac transposes only during chromosome replication, and does not leave a copy behind. There are two possible results of Ac transposition, depending on whether the target DNA has replicated or not (Figure 20.13). - (1) If Ac transposes during replication into a replicated target site, its chromatid’s donor site will be empty since that copy of Ac has inserted elsewhere. In the homologous donor site on the other chromatid, a copy will remain. There is no net increase in copies of Ac. (2) Transposition to an unreplicated chromosome site also leaves one donor site empty (and the other with a copy of Ac). The DNA into which Ac inserts will then be replicated, resulting in a net gain of one copy of Ac.viii. Replication of Ds is the same, except that the transposition protein is supplied by an integrated 20 slide 76 Chapter Ac element.
    77. 77. Fig. 20.12 The structure of the Ac autonomous transposable element of corn and of several Ds nonautonomous elements derived from Ac Chapter 20 slide 77Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
    78. 78. Fig. 20.13 The Ac transposition mechanism Chapter 20 slide 78Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
    79. 79. 5. In Mendel’s wild-type (SS) peas the starch grains are large and simple, while in wrinlded peas (ss) they are small and fissured. a. SS seeds contain more starch and less sucrose than ss seeds. b. The sucrose difference makes ss seeds larger, with higher water content, so that when dried they are wrinided. c. One type of starch-branching enzyme (SBEI) is missing in ss plants, reducing their starch content. d. The SBEI gene corresponding to the s allele has a 0.8 kb transposon similar to the Ax/Ds family inserted into the wild- type S allele. Chapter 20 slide 79
    80. 80. Ty Elements in Yeast1. Ty elements share characteristics with bacterial transposons: a. Terminal repeated sequences. b. Integration at non-homologous sites. c. Generation of a target site duplication (5 bp).2. Ty element is diagrammed in Figure 20.14: a. It is 5.9 kb including 2 terminal direct repeats of 334 bp, the long terminal repeats (LTR) or deltas (δ). b. Each delta contains a promoter and transposase recognition sequences. c. Ty elements encode one 5.7 kb mRNA beginning at the delta 5’ promoter (Figure 20.14). d. There are two ORFs in the mRNA, designated TyA and TyB, encoding two different proteins. e. Ty copy number varies between yeast strains, with an average of about 35.3. Ty elements also share similarities with retroviruses, ssRNA viruses that replicate via dsDNA intermediates. a. Ty elements transpose by making an RNA copy of the integrated DNA sequence, them making DNA using reverse transcriptase. This DNA can integrate at a new chromosomal site. Evidence for this includes: i. An experimentally introduced intron in the Ty element (which normally lacks introns) was monitored through transposition. The intron was removed, indicating an RNA intermediate. ii. Ty elements encode a reverse transcriptase. iii. Virus-like particles containing Ty RNA and reverse transcriptase activity occur. Chapter 20 slide 80 b. Ty elements are referred to as retrotransposons.
    81. 81. Fig. 20.14 The Ty transposable element of yeast Chapter 20 slide 81Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
    82. 82. Drosophila transposons1. It is estimated that 15% of the Drosophila genome is mobile! These transposons fall into different classes: a. The copia retrotransposons include several families, each highly conserved and present in 5-100 widely scattered copies per genome (Figure 20.15). i. All copia elements in Drosophila can transpose, and there are differences in number and distribution between fly strains. ii. Structurally, copia elements are similar to yeast Ty elements: (1) Direct LTRs of 276 bp flank a 5 kb DNA segment. (2) The end of each LTR has 17 bp inverted repeats. (3) An RNA intermediate and reverse transcriptase are used for transposition. (4) Virus-like particles (VLPs) occur with copia. (5) Integration results in target site duplication (3-6 bp). Chapter 20 slide 82
    83. 83. Fig. 20.15 Structure of the transposable element copia, a retrotransposon found in Drosophila melanogaster Chapter 20 slide 83Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
    84. 84. b. P elements cause hybrid dysgenesis, a series of defects (mutations, chromosomal aberrations and sterility) that result from crossing certain Drosophila strains (Figure 20.16). i. A mutant lab strain female (M) crossed with a wild-type male (P) will result in hybrid dysgenesis. ii. A mutant lab strain male (M) crossed with a wild-type (P) female (reciprocal cross) will have normal offspring. iii. Thus, hybrid dysgenesis results when chromosomes of the P male parent enter cytoplasm of an M type oocyte, but cytoplasm from P oocytes does not induce hybrid dysgenesis. Chapter 20 slide 84
    85. 85. Fig. 20.16 Hybrid dysgenesis, exemplified by the production of sterile flies Chapter 20 slide 85Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
    86. 86. iv. The model is based on the observation that the M strain has no P elements, while the haploid genome of the P male has about 40 copies. (1) P elements vary from full-length autonomous elements through shorter versions resulting from a variety of internal deletions. (2) P element transposition is activated only in the germ line. (3) The F1 of an M female crossed with a P male have P elements inserted at new sites, flanked by target site repeats. (4) P elements are thought to encode a repressor protein that prevents transposase gene expression, preventing transposition. (5) Cytoplasm in an M oocyte lacks the repressor, and so when fertilized with P-bearing chromosomes, transposition occurs into the maternal chromosomes, leading to hybrid dysgenesis.v. P elements are used experimentally to transfer genes into the germ line of Drosophila embryos. For example (Figure 20.18): (1) The wild-type rosy (ry) gene was inserted into a P element, cloned in a plasmid and microinjected into a mutant ry/ry strain. (2) Insertion of the recombinant P element into the recipient chromosome introduced the ry allele, and produced wild-type flies. Chapter 20 slide 86
    87. 87. Fig. 20.17 Structure of the autonomous P transposable element found in Drosophila melanogaster Chapter 20 slide 87Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
    88. 88. Fig. 20.18 Illustration of the use of P elements to introduce genes into the Drosophila genome Chapter 20 slide 88Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
    89. 89. Human Retrotansposons1. Retrotransposons also appear to be present in mammals. For example, a very abundant human SINE repeat (short interspersed sequence) is the Mu family, named for the AluI restriction site in its sequence. a. Mu sequences are about 300 bp, repeated 300,000-500,000 times in the human genome (up to 3% of total human DNA). b. Sequences are divergent, related but not identical. c. Each Mu sequence is flanked by 7-20 bp direct repeats. d. At least a few Mu sequences can be transcribed, and the model is that transcriptionally active Mu sequences are retrotransposons that move via an RNA intermediate. e. A human case of a genetic disease, neurofibromatosis, provides some evidence. i. Neurofibromas (tumorlike growths on the body) result from an autosomal dominant mutation. ii. In a patient’s DNA, an unusual Mu sequence was detected in one of the introns of the neurofibromatosis gene. iii. The resulting longer transcript is incorrectly proessed, removing an exon from the mRNA and producing a nonfunctional protein. iv. Neither parent had this Mu sequence in the neurofibromatosis gene. v. Divergent Mu sequences made it possible to track this particular version to an insertion event in the germ line of the patient’s father. f. It is not clear how the functions needed for Mu retrotransposition are provided. Chapter 20 slide 89
    90. 90. 2. A mammalian LINEs family, LINEs-i (Li elements) is also thought to be retrotransposons. a. Humans have 50,000-100,000 copies of the Li element, comprising about 5% of the genome. b. The full-length element (6.5 kb) is not abundant, and most Li elements are deleted versions. c. The full-length Li element contains a large ORF with homolegy to known reverse transcriptases. Experimentally, the Li ORF can substitute for the yeast Ty reverse transcriptase gene. d. Li elements are thought to be retrotransposons, but do not have LTRs. e. Clinically, cases of hemophilia have been shown to result from newly transposed Li insertions into the factor VIII gene. (Factor VIII is required for normal blood clotting.) Chapter 20 slide 90
    91. 91. 21.1 Introduction 91 Figure 21.1
    92. 92. 21.2 Insertion SequencesAre Simple Transposition Modules An insertion sequence is a transposon that codes for the enzyme(s) needed for transposition flanked by short inverted terminal repeats. 92
    93. 93.  The target site at which a transposon is inserted is duplicated during the insertion process. – This forms two repeats in direct orientation at the ends of the transposon.  The length of the direct repeat is: – 5 to 9 bpFigure 21.2 93
    94. 94. 21.3 Composite Transposons Have IS Modules Transposons can carry other genes in addition to those coding for transposition. Composite transposons have a central region flanked by an IS element at each end. 94
    95. 95.  Either one or both of the IS elements of a composite transposon may be able to undertake transposition. A composite transposon may transpose as a unit. – An active IS element at either end may also transposeFigure 21.3 95
    96. 96. by Both Replicative and Nonreplicative Mechanisms  All transposons use a common mechanism in which: – staggered nicks are made in target DNA – the transposon is joined to the protruding 96 ends – the gaps are filled Figure 21.5
    97. 97.  The order of events and exact nature of the connections between transposon and target DNA determine whether transposition is: – replicative – nonreplicativeFigure 21.6 Figure 21.7 97
    98. 98. 21.5 Transposons Cause Rearrangement of DNA Homologous recombination between multiple copies of a transposon causes rearrangement of host DNA. Homologous recombination between the repeats of a transposon may lead to precise or imprecise excision. 98
    99. 99. 21.6 Common Intermediates for Transposition  Transposition starts by forming a strand transfer complex. – The transposon is connected to the target site through one strand at each end. 99Figure 21.11
    100. 100.  The Mu transposase forms the complex by: – synapsing the ends of Mu DNA – followed by nicking – then a strand transfer reaction Replicative transposition follows if the complex is replicated. 100Figure 21.12
    101. 101. 21.7 ReplicativeTransposition Proceeds through a Cointegrate  Replication of a strand transfer complex generates a cointegrate: – A fusion of the donor and target replicons.  The cointegrate has two copies of the transposon. – They lie between the 101 Figure 21.13
    102. 102.  Recombination between the transposon copies regenerates the original replicons, but the recipient has gained a copy of the transposon. The recombination reaction is catalyzed by a resolvase coded by the transposon. 102
    103. 103. 21.8 Nonreplicative Transposition Proceeds by Breakage and Reunion Nonreplicative transposition results if: – a crossover structure is nicked on the unbroken pair of donor strands and – the target strands on either side of the transposon are ligated 103 Figure 21.15
    104. 104.  Two pathways for nonreplicative transposition differ according to whether: – the first pair of transposon strands are joined to the target before the second pair are cut (Tn5), or – whether all four strands are cut before joining to the target (Tn10) 104
    105. 105. 21.9 TnA Transposition Requires Transposase and Resolvase Replicative transposition of TnA requires: – a transposase to form the cointegrate structure – a resolvase to release the two replicons The action of the resolvase resembles lambda Int protein. It belongs to the general family of topoisomerase-like site-specific recombination reactions. – They pass through an intermediate in which the 105
    106. 106. 21.10 Transposition of Tn10 Has Multiple Controls Multicopy inhibition reduces the rate of transposition of any one copy of a transposon when other copies of the same transposon are introduced into the genome. Multiple mechanisms affect the rate of transposition. 106 Figure 21.21
    107. 107. Elements in Maize Cause Breakage and Rearrangements Transposition in maize was discovered because of the effects of chromosome breaks. – The breaks were generated by transposition of “controlling elements.” The break generates one chromosome that has: – a centromere – a broken end 107 – one acentric fragment
    108. 108.  The acentric fragment is lost during mitosis; – this can be detected by the disappearance of dominant alleles in a heterozygote.Figure 21.23 108
    109. 109.  Fusion between the broken ends of the chromosome generates dicentric chromosomes. – These undergo further cycles of breakage and fusion. The fusion-breakage- bridge cycle is responsible for the occurrence of somatic variegation. 109Figure 21.24
    110. 110. 21.12 ControllingElements Form Families of Transposons  Each family of transposons in maize has both autonomous and nonautonomous controlling elements. 110 Figure 21.25
    111. 111.  Autonomous controlling elements code for proteins that enable them to transpose. Nonautonomous controlling elements have mutations that eliminate their capacity to catalyze transposition. – They can transpose when an autonomous element provides the necessary proteins. Autonomous controlling elements have changes of phase, when their properties alter as a result of changes 111
    112. 112. 21.13 Spm Elements Influence Gene Expression Spm elements affect gene expression at their sites of insertion, when the TnpA protein binds to its target sites at the ends of the transposon. Spm elements are inactivated by methylation. 112
    113. 113. 21.14 The Role of Transposable Elements in Hybrid Dysgenesis P elements are transposons that are carried in P strains of Drosophila melanogaster, but not in M strains. When a P male is crossed with an M female, transposition is activated. 113
    114. 114.  The insertion of P elements at new sites in these crosses: – inactivates many genes – makes the cross infertile 114 Figure 21.28
    115. 115. 21.15 P Elements AreActivated in the Germline  P elements are activated in the germline of P male x M female crosses.  This is because a tissue-specific splicing event removes one intron. – This generates the coding sequence for Figure 21.29 115
    116. 116.  The P element also produces a repressor of transposition. – It is inherited maternally in the cytoplasm.  The presence of the repressor explains why M male x PFigure 21.30 female crosses remain fertile. 116
    117. 117. Pray, L. (2008) Transposons: Thejumping genes. NatureEducation 1(1)
    118. 118. DNA transposons Seen in both prokaryotes and eukaryotes – the IS element (insertion sequence) in bacteria – DNA transposons seen in eukaryotic genomes (P elements in fruit flies, Ac/Ds elements in plant genomes) Mechanism of transposon action – Transposon encodes an enzyme: transposase – Transposase excises itself out and then is able to cut in the middle of a target DNA – Effect is based on where the transposable
    119. 119. RNA transposable elements Derived from an RNA intermediate Seen only in eukaryotic genomes Originated from ancient retroviral genome – Retrotransposon  LTR elements – Retroposons  SINE-human  LINE-human
    120. 120. - Derived from a viral genome from the retrovirus: LTR gag RT env LTR RT: reverse transcriptase ~7 kb LTR: long terminal repeat gag, env: encode proteins needed for retroviral assembly and infectionRetroelements: missing some or most of the complete retroviralgenome;
    121. 121. - Retrotransposons:contain the LTR repeats LTR gag RT LTR ~7 kb -make up ~50% of the maize genome
    122. 122. Mechanism of retrotransposition RNARetrotransposon Transcription Reverse transcription RNA DNARetrotransposon Retrotransposon copy
    123. 123. Human Retroposons: non-LTR- LINE: long interspersed elements gag? RT poly(A) ~6 kb-SINE: short interspersed element;The Alu element is a well known example poly(A) ~0.3 kb
    124. 124. C-value paradox: genome size notalways predictor of gene numberTaken fron
    125. 125. Transposable Elements DNA Sequences That Change Positions in the Genome
    126. 126. Types of Transposable ElementsType Transposition ExamplesTransposon Use a DNA Corn: Ds element(Class I) intermediate Drosophila: P elementRetrotransposons Use an RNA Yeast: Ty(Class II) intermediate Drosophila: Copia Human: Alu Human: L1Transposition: movement of a transposable element
    127. 127. Characteristics of Transposable Elements All elements have direct repeats: short repeated sequences flanking the element, arise by transposition
    128. 128. Characteristics of Transposable Elements Some elements have terminal inverted repeats
    129. 129. Characteristics of Transposable Elements  Carry gene for enzyme that catalyzes transposition – transposase for elements that use a DNA intermediate – reverse transcriptase for elements that use an RNA intermediate  May contain other genes
    130. 130. Mechanisms of Transposition Use of a DNA Intermediate – Replicative- new copy in new location, old copy retained at original site, element is used as template to produce the new copy
    131. 131. Mechanisms of Transposition Use of a DNA Intermediate – Non-replicative: moves to another site without replication of the element
    132. 132. Mechanisms of Transposition Use of an RNA Intermediate – element is transcribed – reverse transcriptase produces a double-stranded DNA copy for insertion at another site
    133. 133. Types of Retrotransposons Viral Retrotransposons – resemble retroviruses = viruses with an RNA genome  Long terminal direct repeat at each end  Carry genes for enzymes usually found in RNA viruses
    134. 134. RetrovirusCharacteristic s
    135. 135. Types of Retrotransposons Non-viral Retrotransposons – do not resemble retroviruses – two types in humans  LINES = long interspersed elements – 6-7 kb long – example: L1 has 600,000 copies, represents 15% of human DNA  SINES = short interspersed elements – 300 bp long – example: Alu has 1 million copies, represents 10% of human DNA
    136. 136. Definitions and Keywords Transposons - are sequences of DNA that can move around to different positions within the genome of a single cell, a process called transposition. Transposase -An enzyme that binds to ends of transposon and catalyses the movement of the transposon to another part of the genome by a cut and paste mechanism or a replicative transposition mechanism. IS elements -A short DNA sequence that acts as a simple transposable element
    137. 137. Definitions and KeywordsDNA polymerase-A DNA polymerase is an enzyme that catalyzes the polymerization of deoxyribonucleotides into a DNA strand.DNA ligase is a special type of ligase that can link together two DNA strands that have double-strand break a break in
    138. 138. Bacterial Transposons Bacteria contain two types of transposons 1]Composite mobile genetic elements that are larger than IS elements and contain one or more protein-coding genes in addition to those required for transposition. 2]Non composite mobile genetic elements are those which lack IS elements on its ends e.g. is Tn3
    139. 139. Transposone Presented by
    140. 140. Transposable ElementsThey are discrete sequence in thegenome that are mobilethey are able to transport themselvesto other location. Other names: Jumping genes Selfish DNAs Molecular parasites Controlling elements TEs are present in the genome all species of three domains
    141. 141. What do we want to know about mobile genetics elements? 1 – The history of mobile genetic elements 2 – The classification of TEs 3 – The structure of TEs 4 – The mechanism of transposition 5 – The effects of TEs on gene and genome 6 – The use of TEs as molecular
    143. 143.  TRANSPOSONS “Transposable elements” “Jumping genes” Mobile DNA – able to move from one place to another within a cell’s genome – sometimes a copy is made and the copy moves – insertion requires target DNA sequences
    144. 144. Transposon inverted terminal repeat(ITR)
    145. 145.  In the process, they may - cause mutations. - increase (or decrease) the amount of DNA in the genome. - promote genome rearrangements. - regulate gene expression. - induce chromosome breakage and
    146. 146. Discovery of transposons Barbara McClintock 1950’s Ac Ds system in maize influencing kernel color unstable elements changing map position promote chromosomal breaks. Rediscovery of bacterial insertion sequences source of polar mutations discrete change in physical length of DNA inverted repeat ends: form “lollipops” in EM after denaturation.
    147. 147. These mobile segments of DNA are sometimes called "jumping genes" There are two distinct types of transposons: 1) DNA transposons -transposons consisting only of DNA that moves directly from place to place 2) Retrotransposons - first transcribe the DNA into RNA and then - use reverse transcriptase to make a DNA copy of the RNA to insert in a new location
    148. 148. Classification of Transposons into two classes In both cases ds DNA intermediate is integrated into the target site in DNA to complete
    149. 149. BACTERIAL TRANSPOSONS In bacteria, transposons canjump from chromosomal DNA toplasmid DNA and back. Transposons in bacteria usuallycarry an additional gene forfunction other than transposition---often for antibiotic resistance. Bacterial transposons of thistype belong to the Tn family. Whenthe transposable elements lackadditional genes, they are known
    150. 150. BACTERIAL TRANSPOSONS - TYPES1. Insertion sequence2.Composite transposon3.Tn3-type transposon4.Transposable phage
    151. 151. 1.Insertion sequencesInsertion sequences – IS1 and IS186, present in the 50-kb segment of the E. coli DNA, are examples of DNA transposons. Single E. coli genome may contain 20 of them.Most of the sequence is taken by one or two genes for transposase enzyme that catalyses transposition. IS elements transpose either replicatively
    152. 152. cont….IS elementsStudy of E. coli mutations resulting from insertion of 1-2 kb longsequence in the middle of certain genes.Inserted stretches or insertion sequences – could bevisualized by EM. IS - molecular parasites in bacterial cells.Transposition of IS is very rare – one in 105-107 cellsper generation. Higher rates result in greater mutation rates.
    153. 153. Bacterial IS element Central region encodes for one or two enzymes required for transposition. Itis flanked by inverted repeats of characteristic sequence.The 5’ and 3’ short direct repeats are generated from the target-site DNAduring the insertion of mobile element.The length of these repeats is constant for a given IS element, but theirsequence depends upon the site of insertion and is not characteristic for theIS element.Arrows indicate orientation.
    154. 154. Insertion sequences in E.coliElements Size (bp) No.of.copies/ genomeIS1 768 8IS2 1327 5IS3 1300 1 or moreIS4 1426 1 or more
    155. 155. 2.Composite transposons Bacteria contain composite mobilegenetic elements that are larger than ISelements and contain one or moreprotein-coding genes in addition to thoserequired for transposition: Composite transposons - are basicallythe pair of IS elements flanking asegment of DNA usually containing one ormore genes, often coding for ABresistance.
    156. 156. Cont…2.Composite transposon - Antibiotic resistant gene - Flank by IS element (inverted or directed repeat) - Terminal IS can transpose by in self Ex. Tn5, Tn9, Tn10
    157. 157. 3. Tn 3 transposon family - 5000 bp - code for Transposase, β-lactamase, Resolvase - Function of resolvase Decrease Transposaseproduction Catalyse therecombination of
    158. 158. Cont… ITRITR transposase resolvase β-lactamase  Tn3 – type transposon --- 5kb  ITR - inverted terminal repeat  β- lactamase – Resistance gene
    159. 159. 4.Transposable phage Transposable phages – bacterial viruses which tranpose replicatively as a part of their normal infectious cycle. Integrate into E. coli chromosome at regulatory element
    160. 160. Transposable phage ITRITR Integration and Protein coat Lysis genes Replication genes genes  Transposable phage – 38kb  ITR - inverted terminal repeats
    161. 161. Transposable phage - Mu phage
    162. 162. Mechanism of transpositionTwo distinct mechanisms oftransposition: Replicative transposition – directinteraction between the donortransposon and the target site,resulting in copying of the donorelement Conservative transposition –
    163. 163. Mechanism of transposition 1. Replicative transposition Copy of transposon sequenceTransposase enzyme cut target DNA Transposition Duplication of target sequence
    164. 164. Replicative transposition
    165. 165. 2. Non-replicative (conservative) transposition- Cannot copy transposon sequence- Transposition by cut and paste model Cut transposon sequence from donor molecule attach to target site
    166. 166. Non-replicative (conservative) transposition
    167. 167. Mechanism oftransposition
    168. 168. Evolution of Transposons Transposons are found in all major branches of life. It arisen once and then spread to other kingdoms by horizontal gene transfer. Duplications and DNA rearrangements contributed greatly to the evolution of new
    169. 169. Cont… Mobile DNA most likely also influenced the evolution of genes that contain multiple copies of similar exons encoding similar protein domains (e.g., the fibronectin gene). The evolution of an enormous variety of antibiotic resistance transposons and their spread among bacterial species. example of genetic adaptation
    170. 170. Transposons causing diseases Transposons are mutagens. They can damage the genome of their host cell in different ways: 1. A transposon or a retroposon that inserts itself into a functional gene will most likely disable that gene. 2.After a transposon leaves a gene, the resulting gap will probably not be repaired correctly. 3.Multiple copies of the same sequence, such as Alu sequences can hinder precise chromosomal pairing during mitosis and meiosis, resulting in unequal crossovers, one of the main reasons for chromosome duplication.
    171. 171. Cont… Diseases caused by transposons include -hemophilia A and B -severe combined immunodeficiency -Porphyria -Cancer
    172. 172. Applications The first transposon was discovered in the plant maize (Zea mays, corn species), and is named dissociator (Ds). Likewise, the first transposon to be molecularly isolated was from a plant (Snapdragon). Transposons have been an especially useful tool in plant molecular biology. Researchers use transposons as a means of mutagenesis.
    173. 173. Cont… To identifying the mutant allele. To study the chemical mutagenesis methods. To study gene expression. Transposons are also a widely used tool for mutagenesis of most experimentally tractable
    174. 174. QUERIES?
    175. 175. Why study mobile genetic elements? They are the major forces driving evolution They can cause genome rearrangement (mutation , deletion and insertion ) They have wide range of application potentials
    176. 176. The discovery of mobile genetic elements Transposable elements Phage Plasmid DNA
    177. 177. The discovery of transposable elements Barbara Mc Clintock discovered TEs in maize (1983) Her work on chromosome breakage began by investigating genetic instability (1983) Observing variegated patterns of pigmentation in maize plant and kernels New kinds of genetic instability She spent the next tree decades for this genetic elements Controlling elements (1956)
    178. 178. Barbara Mc Clintock 1902 1980 ( noble in 1984)
    179. 179. Plasmid , phage Cell to cell conjugation Bactriophage mediated transduction Bill Hayes ( 1952 ) Ellin Wollman and Francois Jancob , 1961 Alan Campbell
    180. 180. Classification of transposable elements DNA transposons Retrotransposons
    181. 181. Autonomous and non autonomous elements Both class are subdivided into distinct superfamilies and families Structure feature , internal organization , the size of target site duplication , sequence similarities at the DNA and protein levels Autonomous : they have the ability to excise and transpose non autonomous elements- They don’t transpose- They become unstable only when an autonomous member of same family is present elsewhere in the genome- They are derived from autonomous elementsA family consists of single type of autonomous element accompanied by many varieties of non autonomous elements
    182. 182. DNA based elements Insertion sequence (IS) The simplest (smallest) transposons are called IS The IS elements are normal constituents of bacterial chromosome and plasmids Spontaneous mutation of the lac and gal operons They are autonomous units ,each of which codes only transposase
    183. 183. Structure of IS
    184. 184. Composite transposone One class of large transposons are called Composite transposons They carring the druge marker is flanked on either side by arms that consist of IS elementsIS modules- identical (bothfunctional: Tn9; Tn903) orclosely related (differ infunctional ability: Tn10; Tn5) 1. A functional IS module can transpose either itself or the entire transposon
    185. 185. Mechanism of transpositionDirect repeats aregenerated byintroduction ofstaggered cuts whoseprotruding ends arelinked to the transposon. The stugger between the cuts determines the length of the direct repeats. The target repeat is characteristic of each transposon; reflects the
    186. 186. Mechanism of transposition 1- Replicative transpositon1. Replicative :a) Transposon is duplicated; a copy of the original element is made at a recipient site(TnA); donor keeps original copyb) Transposition- an increase in the number of Tn copiesc) ENZs: transposase (acts on the ends of original Tn) and resolvase (acts on the duplicated copies)
    187. 187. Mechanism of transposition 2 -Nonreplicative Nonreplicative : Transposon moves from one site to another and is conserved; breaks in donor repaired b) IS and Tn10 and Tn5 use this mechanism; no Tn copy increase c) ENZs: only transposase
    188. 188. Donor cut  The first stages of Both replicative and non-replicative transpositio are semilar  IS elements, prokaryotic eukaryotic transposons, and1. Synapsis stage- two .ends of bacteriophage Mutransposon are brought together 2. Transposon nicked at both ends; target nicked at both strands 3.. Nicked ends joine crosswise;covalent connection between the transposon the target
    189. 189. Mu integrates by nonreplicative transposition; during lyticcycle- number of copies amplified by replicativetransposition 22 bp - MuA binds to ends as tetramer forming a synapsis. - MuA subunits act in trans to cut next to R1 and L1 (coordinately; two active sites to manipulate cuts in transfers trans in trans DNA). - MuA acts in trans to cut the target site DNA and mediate in trans strand transfer
    190. 190. The chemistry of Donor and target cut OH OH OHCutting of end only OH  Cutting of both ends The 3’-ends ends groups released from flanking DNA by donor cut reaction They are nuclophile that attack phosphodiester bonds in target DNA
    191. 191.  The product of these reaction is strand transfer complex transposon target In strand transfer complex transposon is connected to the target site through one strand at each end Next step differs and determines the type of transposition: Strand transfer complex can be target for replication (replicative transposition) or for repair (nonreplicative transposition; breakage & reunion) Strand transfer complex
    192. 192. Molecular mechanism of transposition (I) Replicativetransposition Replicative transposition proceeds through a cointegrate.Transposition may fuse a donor andrecipient replicon into a cointegrate.Resolution releases two replicons-eachhas copy of the transposon
    193. 193. transposition Donor and target cut Ligation to target ends3. 3’-ends prime replicationThe crossover structure contains a single strandedregion at each of the staggered ends=pseudoreplication forks that provide template forDNA synthesis cointegrate.
    194. 194. additional nickingcommonstructure Non-replicative Breakage & reunion Replicative
    195. 195. Retrotransposon ( retroposons ) Use of an RNA Intermediate – element is transcribed – reverse transcriptase produces a double-stranded DNA copy for insertion at another site – they as other elements
    196. 196. Types of Retrotransposons1 – viral superfamily (autonomousretrotransposon) – retrovirus – LTR- retrotransposon – LINES non LTR- retrotransposon2 – nonviral superfamily (non autonomous retransposons) SINES
    197. 197. retrovirus RNAreverstranscriptase Liner DNA Integration provirus Transcription RNA
    198. 198. LTR - retrotrasposon pol Reverse transcriptase (RT) Integrase (IN) Ribonuclease H (RH) gag ? env
    199. 199. mechanism of transposition Integrase acts on both the retrotransposon line DNA and target DThe integrase bring the ends of the linear DNA together 5’-ends-Generate 2 base recessed 3’ -ends 3’-endsand staggered end in target DNA
    200. 200. Non – LTR retrovirus LINES = long interspersed elements SINES = short interspersed elements don’t terminate in the LTRs they are significant part of relatively short sequence of mammalian genomes .
    201. 201. Effect of transposabli elements on gene and genome TEs cause a varity of change in the genome of their hosts this ability to induce mutation depend on their of capability of transposing some arrangement can be beneficial they can advantageous for adaptation to new environment play important role in evolution . they have the ability to rearrange genomic information in several ways1 – Modification of gene expression2 – Alternation gene sequence3 – Chromosomal structural changes
    202. 202. Modification of gene expression insertion of a TE within or adjacent to a gene the element blocks or alters the pattern of transcription . insertion of Fot1 in a intron of niad (F . oxysporum ) different mutant transcripts all were shorter They result from:
    203. 203. Alternation gene sequence cut and pate mechanism often produce variation when they excise . the excision process may result in addition of a few base pair ( footprint ) at donor site . these footprint cause diversification of nucleotide sequence and new functional alleles Example :Fot1 and Impala generally leave 4 bp – ( 108 ) or 5 – ( 63 ) foot prints
    204. 204. Chromosomal structural changes TEs can produce a series of genome rearrangment through ectopic recombination deletion , duplication , inversion and translucation mediate by TEs ( Drosophila , Yeast , human ) karyoptypic variation in natural isolate in fungai high level of chromosome – length polymorphism (Magnoporthe grisea , F. oxysporum) translocation tox1 of Cochliobolus
    205. 205. Use of TEs as molecular tools Use as strain specific diagnostic tools TEs are often restricted to specific strains identify specific pathogen in plant pathology Fot1 ( F. oxysporum f sp. albedians ) provide PCR targets a sensitive detection thechnique to prevent the introduction of pathogenic form
    206. 206. Use of TEs as molecular tools Tools for the analysis of population structure MGR 586 ( Magneporthe grisea ) oryza : 30 – 50 wheat and other ( 1 – 2 ) they have used to distinguish genetically divergent population fingerprinting of isolates
    207. 207. Gene taggingas molecular tools Use of TEs with transposable elementsjumping into coding region Disrupt target gene Target gene can easily determined by PCR arise mutant methods phenotype
    208. 208. Thanks for attention
    209. 209. A composite transposon, is flanked by two Composite Transposonseparate IS elements which may or may notbe exact replicas. Instead of each IS elementmoving separately, the entire length of DNAspanning from one IS element to the other istransposed as one complete unit. IR IR
    210. 210. Non composite Transposon Non-composite transposons (which lack flanking insertion sequences). In each case, transposition requires specific DNA sequences located at the ends (IS1, IS3, Tn5, Tn10, and Tn3) or a multisubunit complex (e.g. Tn7). Encode transposition proteins, have inverted repeats (but no ISs) at their ends. In addition to resistance and virulence genes they may encode catabolic enzymes
    211. 211. Mechanism of transposition There are two mechanisms of transposition replicative and nonreplicative During transposition, the IS-element transposase makes cuts at the positions indicated by small red arrows, So the entire transposon is moved from the donor DNA (e.g., a plasmid). A DNA polymerase fills in the resulting gaps from the sticky ends and DNA ligase closes the sugar-phosphate backbone. This results in target site duplication and the insertion sites of DNA transposons may be identified by short direct repeats (a staggered cut in the target DNA filled by DNA polymerase) followed by inverted repeats (which are important for the transposon excision by transposase). The duplications at the target site can result in gene duplication and this is supposed to play an important role in evolution. Composite transposons will also often carry one or
    212. 212. Mechanism of transposition(contd)The conservative mechanism, also called the “cut-and-paste” mechanism, is used by elements like Tn10 . The element is excised cleanly by double-strand cleavages from the donor DNA and inserted, with limited repair, between a pair of staggered nicks at the target DNA.Replicative transposition is a mechanism of transposition in molecular biology in which the transposable element is duplicated during the reaction, so that the transposing entity is a copy of the original element. Replicative transposition is characteristic to retrotransposons and occurs from time to time in class II transposons.Retrieved from "
    213. 213. General mechanism of TranspositionProduction of protein (enzyme transposase) from the site oftransposase(right corner an Tn 5) should be shown.{the site in upperdiagram in between IR of IS element.}Action/Motion-Production of protein (enzyme transposase) from the siteof
    214. 214. ReplicativeTransposition Single stranded cuts are made on either side of the Transposon and on the opposite sides of the target of the recipient.
    215. 215. This produces 4 free ends get in each DNA molecule Two of the ends from the donor are ligated to 2 of the ends of target. This links the two molecules with a single molecule of transposon.
    216. 216. The two remaining free 3’ ends areused as primers for DNA polymerasewhich uses the Transposon DNAas the template.This replicates thetransposon and leaves the cointegrate.
    217. 217. NickingSingle strranded cuts produce staggered ends in both transposon and
    218. 218. Crossover structure (strand transfer complex)Nicked ends of Transposon are joined to nicked ends of target.
    219. 219. Replication from free 3’ end generate cointegrateSingle molecule has two types of transposon.
    220. 220. Cointegrate drawn as continuous path shows that transposonsare at junctions between replicons.
    222. 222. First, the transposase makes a double-stranded cut in the donor DNA at the ends of the transposonand makes a staggered cutin the recipient DNA.
    223. 223. Each end of the donor DNA is thenjoined to an overhangingend of the recipient DNA.
    224. 224. DNA polymerase fills in the short,overhanging sequences,resulting in a short, direct repeaton each side of the transposoninsertion in the recipient DNA.
    225. 225. INSTRUCTIONS SLIDE1 Questionnaire to test the userQ1]Define tranposition?Transposons sequences DNA2 the genome ofaresingle cell, ofprocessthat can move around to different positions within a a called transposition.Q2]Give examples of non composite transposons.IS1, IS3, Tn5, Tn10, and Tn3) or a multisubunit complex (e.g. Tn7)Q3]Describe the general structure of bacterial transposons.3 Ans45
    226. 226.  This transposon consists of a chloramphenicol-resistance gene (dark blue) flanked by two copies of IS1 (orange), one of the smallest IS elements. Other copies of IS1, without the drug- resistance gene, are located elsewhere in the E. coli chromosome. The internal inverted repeats of IS1 abutting the resistance gene are so mutated that transposase does not recognize them. During transposition, the IS-element transposase makes cuts at the positions indicated by small red arrows, so the entire transposon is moved from the donor DNA (e.g., a plasmid). The target- site sequence at the point of insertion becomes duplicated on either side of the
    227. 227.  Q4]Explain the mobile genetic elements found in bacteria.ANS:-Three of the many types of mobile genetic elements found in bacteria. Each of these DNA elements contains a gene that encodes a transposase, an enzyme that conducts at least some of the DNA breakage and joining reactions needed for the element to move. Each mobile element also carries short DNA sequences (indicated in red) that are recognized only by the transposase encoded by that element and are necessary for movement of the element. In addition, two of the three mobile elements shown carry genes that encode enzymes that inactivate the antibiotics ampicillin (ampR) and tetracycline (tetR). The transposable element Tn10, shown in the bottom diagram, is thought to have evolved from the chance landing of two short mobile elements on either side of a tetracyclin-resistance gene; the wide use of tetracycline as an antibiotic has aided the spread of this gene through bacterial populations. The three mobile elements shown are all examples of DNA-only transposons
    228. 228.  Q5]Illustrate the mechanism of transposition in transposons. ANS:-
    229. 229. 1 Links for further reading2 Molecular Cell BiolOGY Baltimore -molecUlar biology of the gene watson -Genes Lewin -VOET AND VOET3 -LEHNINGER -COOPER45
    230. 230. Thank you
    231. 231. Applying Your Knowledge 1. Retrotransposon 2. Transposon 3. Both retrotransposons and transposons 4. Neither retrotransposons nor transposonsWhich type of transposable element• Uses a DNA intermediate for transposition?• Contains long terminal repeats on its ends?• Generates direct repeats as a result of transposition?• Carries a gene for reverse transcriptase?• Can insert a copy in a new location while leaving the old copy at the original site?
    232. 232. Effects of TranspositionTransposable elements can: Cause mutations in adjacent genes Cause chromosomal rearrangements Relocate genes
    233. 233. Possible Advantages of Transposable Elements Transposable elements may:  Create genetic diversity  Act as promoters  Allow recombination between plasmid and genomic DNA when multiple copies of the element are present  Carry antibiotic resistance genes, conferring an advantage on bacterial cells  Increase the number of copies of an exon or gene
    234. 234. Examples of Transposable Elements Bacterial Insertion Sequences and more Complex Transposons Ac-Ds Elements in Corn P elements in Fruit Flies
    235. 235. Transposable Elements in Bacteria Insertion Sequences contain only the elements needed for transposition Composite Transposons contain DNA that has insertion sequences on both sides Antibiotic resistance genes are often included
    236. 236. Ac and Ds Elementsin Corn Ac stands for activator element Ds stands for dissociative element Barbara McClintock showed that --transposition of the Ds element altered kernel coloration --movement of the Ds element required the activity of Ac element
    237. 237. Transposition of Ds ElementDisrupts Gene Controlling Kernel Color
    238. 238. Excision of Ds Element Leads to Variegated Kernels
    239. 239. Relatedness of Ac and Ds ElementsFor transposition, Ds elements require thetransposase produced by the Ac element.
    240. 240. Autonomous and Non- autonomous Elements Type Properties ExampleAutonomous •Can transpose without Ac the presence of another elementNon- •Requires the Dsautonomous presence of another functional element to move •Autonomous element provides transposase or reverse transriptase
    241. 241. The P Element in Drosophila Codes for a Transposase and a Repressor of Transposition No Repressor repressor produced P element Transposition is inserts in repressed multiple locations
    242. 242. Use of the P Element As a Vector in Drosophila P element codes for transposaseP element with gene of interest can insert into chromosomeswith help of plasmid containing only transposase.
    243. 243. Applying Your Knowledge 1. Ac-Ds Elements 2. Alu Element 3. Insertion Sequence 4. P elementWhich type of transposable element• Contains only the sequences needed for transposition in bacteria?• Represents a SINE found in humans?• Is used to insert genes into fruit fly chromosomes?• Causes reversible alterations for kernel color in corn?