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Transposon

Transposon. The discovery of Transposon. plant genomes are very rich in transposons. The ability of transposable element to alter the gene expression can often observed as dramatic variation in the coloration of the plant. The transposable elements

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Transposon

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  1. Transposon

  2. The discovery of Transposon • plant genomes are very rich in transposons • The ability of transposable element to alter the gene expression can often observed as dramatic variation in the coloration of the plant • The transposable elements were first discovered in plants by Barbara McClintock in late 1940s

  3. “Transposable element” • DNA sequence able to insert itself (or copy of itself) at a new location in the genome without having any sequence relationship with the target locus

  4. Characteristics of “Transposable element” • discrete sequence in the genome • able to transport themselves to other locations within genome= mobile • it is not independent form like phage or plasmid, but it is in the genome and move directly from one site to other • the insertion does not rely on the relationship of sequence between donor and recipient site • Major source of mutation in the genome

  5. Transposable element has 2 general classes (1) Transposon • sequence of DNA coding for proteins that are directly manipulate DNA to propagate themselves within the genome

  6. Transposable element has 2 general classes (2) Retroposon • relates to retroviruses, it has ability to make DNA copies of their RNA transcripts, the DNA copies then integrated at new site in the genome

  7. Transposon How transposon move? carries genes that code for the enzyme that required for its own transposition Transposase

  8. Transposase enzyme contains 3 domains DDE active site domain • active site for DNA cleavage and DNA strand transfer • it has two aspartate (D) and glutamate (E) • require divalent metal ion (Mg2+ or Mn2+) for activity • this domain is highly conserved for each transposase need for assemble the DNA-protein complex specific for each individual element Site-specific DNA-binding domain Protein-protein interaction region

  9. Transposase enzyme • Transposases are active when assemble into synaptic complex called “transposome” (nucleoprotein) • The target site is selected by the effect of transposase Some choose randomly, Some choose for consensus seq

  10. Types of transposon • Insertion sequence (IS) element • Composite Transposon

  11. Insertion sequence (IS element) • It is a simple transposition module (autonomous unit) • IS is a small bacterial transposon that carries only the genes needed for its own transposition (transposase) • IS is flanked by inverted terminal repeats • Inverted terminal repeats are the short related or identical sequence present in the reverse orientation at the end of transposon

  12. The target site for transposon inserted is duplicated during insertion process to form two repeats in direct orientation at the ends of transposon

  13. How the target site is duplicated? The staggered nicks (single strand nicks) are made in target DNA The transposon is joined to protruding ends gaps are filled to generate a duplicated of target repeats ATGCA TACGT

  14. each type is given prefix IS, followed by a number that identify type • each IS element is different in sequence

  15. Composite Transposon • Bacterial transposons carrying marker gene, such as drug resistance, are named as “Tn” followed by number • Composite transposons have a central region flanked on each side by “arms” that consist of insertion sequences (IS), either one or both of IS may enable the entire element to transpose • “arms” may be either the same IS in the same or different orientation (direct repeat or inverted repeat)

  16. can cause transposition

  17. Mechanism of transposition • Replicative transposition The movement of transposon by a mechanism in which first it is replicated, and one copy is transferred to new site

  18. Mechanism of transposition • Nonreplicative transposition The movement of transposon that leaves a donor site and move to new site

  19. Mechanism of Replicative transposition • Transposable element that use this mechanism for transposition are Phage Mu and Tn3 • Mechanism: replicative transposition proceeds through a cointegrate • enzyme needed: Transposase and Resolvase

  20. Mechanism of Replicative transposition • The process start with the formation of the strand transfer complex (nucleoprotein involves transposase, resolvase and DNA) • The donor and the target strands are nicked to generate the staggered ends in both transposon and target site

  21. Mechanism of Replicative transposition • The donor and target ends are ligated • The strand transfer complex generate a crossover shaped structure (provide pseudoreplication fork start from staggered ends) • transposon is duplicated via the replication and create copies of both target and donor sites

  22. The replication will be terminated at the ends and produce “cointegrate”

  23. The homologous recombination between the two copies will separate the cointegrate into two replicons that contain transposon on both replicon need resolvase • This reaction call “resolution”

  24. Mechanism of Nonreplicative transposition • Transposable element that use this mechanism for transposition are Tn5, Tn10, Tn7, IS911, Tn917 • Mechanism: Proceeds by breakage and reunion allow the target to be reconstructed, while the donor remain broken with double strand break • enzyme needed: Transposase

  25. Mechanism of Nonreplicative transposition • The beginning of reaction similar to replicative transposition • However, the unbroken strand of donor have been nicked, then the target strands on either sides can be ligated

  26. Tn10 • both strands of Tn10 are cleaved and transposon is joined to the nicked target site

  27. Tn5 • Another mechanism of nonreplicative transposition is generated by Tn5 • start with one strand is nicked, then the 3’-OH end attack at the other strand and results in joining of the two strands of transposon as hairpin form • The activated water molecule attacks the hairpin, then it can join with staggered ends of target DNA

  28. Conclusions “The transposable elements can promote rearrangements of the genome by addition, deletion or inversion of the host sequence” http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter13/animation_quiz_5.html

  29. Extrachromosomal DNA

  30. Extrachromosomal DNA is any DNA that is located outside of the nucleus or sometime call cytoplasmic DNA, which also serve important biological functions Plasmid Episome Viral DNA Mitochondrial DNA microDNA (circular DNA) Chloroplast

  31. Extrachromosomal DNA • A bacterium may be a host for independently replicating genetic units in addition to its chromosome • There are two general types; viral DNA and Plasmid

  32. Viral DNA (from bacteriophage)

  33. Require a host to replicate • Viral genomes can be made up of single stranded DNA (ssDNA) • double stranded DNA (dsDNA) • single stranded RNA (ssRNA) • double stranded RNA (dsRNA). • Viruses can also be found in both linear and circular form

  34. Plasmids • plasmids are self-replicating circular molecule of DNA that are maintained in the cell and remains constant from generation to generation • “single copy plasmids” are maintained at the same relative quantity as host chromosome that is one per unit bacterium and segregated equally at each bacterial division • “multicopy plasmids” exist in many copies per unit bacterium and may be segregated to daughter bacteria that always gain some more copies by random distribution

  35. Natural Plasmids Linear bacterial plasmids have been identified in several species of spirochete bacteria, including members of the genus Borrelia

  36. Episomes • The plasmids that have ability to behave integration into chromosome • Related processes are used by phage and episomes to insert into and excise from the bacterial chromosome

  37. Mitochondrial DNA

  38. Human mitochondrial DNA

  39. Chloroplast DNA

  40. Small circles of extrachromosomal DNA (200-400 bp) appear to be widespread in mammals, and may be byproducts of small deletions in the nuclear DNA of somatic cells. microDNA (circular DNA) It’s unclear what processes underlie microDNA formation, but it’s most likely they occur during DNA replication or repair. microDNAs are rich in cytosines and guanines, and tend to cluster at the 5’ untranslated areas, suggests the possibility that nucleosomes important for gene regulation may be involved.  Y. Shibata et al., "Extrachromosomal MicroDNAs and Chromosomal Microdeletions in Normal Tissues," Science, doi:1-.1126/science.1213307, 2012.

  41. Mechanism of extrachromosomal DNA replication (1) Replicative of linear DNA • using the mode of “Strand displacement” in which a new DNA strand grows by displacing the previous (homologous) strand of duplex (found in some viruses: Adenovirus) (2) Replicative of circular DNA

  42. The initiation of replication starts from “a terminal protein” that allows replication of a linear phage genome to start at the end • The protein attaches to the 5’end of the genome through a covalent bond, and associated with a DNA polymerase, and contains cytosine residue that serves as primer

  43. Terminal protein provides a primer

  44. A newly synthesize strand displaces the corresponding strand of the original duplex • The strand that is released will pair at the ends to form a duplex origin that initiates synthesis of a complementary strand

  45. Mechanism of extrachromosomal DNA replication (2) Replicative of circular DNA • “The rolling circle” is the mode of replication in which a replication fork proceeds around a circular template for an indefinite number of revolution

  46. The DNA strand newly synthesized in each revolution displaces the strand synthesized in the previous revolution • giving a tail containing a linear series of sequence complementary to the circular template strand • A rolling circle generates single strand multimers of the original sequence

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