Transposons

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Transposons

• Dr Derakhshandeh

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Mobile Genetic Elements

• Transposons or Transposable elements (TEs)
• move around the genome

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Transposable elements in prokaryotes

• Insertion sequence (IS) elements
• Transposons (Tn)
• Bacteriophage Mu

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Insertion sequence (IS) elements

• Simplest type of transposable element found in
  bacterial chromosomes and plasmids
• Encode only genes for mobilization and insertion
• Range in size from 768 bp to 5 kb
• IS1 first identified in E. colis glactose operon
  is 768 bp long and is present with 4-19 copies in
  the E. coli chromosome
• Ends of all known IS elements show inverted
  terminal repeats (ITRs)

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Integration of IS element in chromosomal DNA
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Three different mechanisms for transposition

• Conservative transposition
• Replicative transposition
• Retrotransposition

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Three different mechanisms for transposition

• Conservative transposition The element itself
  moves from the donor site into the target site
• Replicative transposition The element moves a
  copy of itself to a new site via a DNA
  intermediate
• Retrotransposition The element makes an RNA copy
  of itself which is reversed-transcribed into a
  DNA copy which is then inserted (cDNA)

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Conservative transposition
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Replicative transposition
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Retrotransposition
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common feature of mobile elements

• Generation of short direct repeats flanking the
  newly inserted element
• This results for a staggered cut being made in
  the DNA strands at the site of insertion

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Transposons (Tn)

• Similar to IS elements but are more complex
  structurally and carry additional genes
• 2 types of transposons
• Composite transposons
• Noncomposite transposons

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• Composite transposons

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IS10R is an autonomous element, while IS10L is
non-autonomous
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Composite Transposons

• Tetracycline resistance is carried by a
  transposable element
• The transposon is a composite transposon,
  composed of IS-elements flanking an included
  sequence, in this case containing an antibiotic
  resistance gene
• IS10R is an autonomous element
• while IS10L is non-autonomous
• Composite transposons probably evolved from IS
  elements by the chance location of a pair in
  close proximity to one another. Inactivation of
  one element by mutation would not harm ability to
  transpose and would assure continued
  transposition of the entire transposon

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• Noncomposite transposons

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Noncomposite transposons (Tn)

• Carry genes (e.g., a gene for antibiotic
  resistance)
• Ends are non-IS element repeated sequences
• Tn3 is 5 kb with 38-bp ITRs and includes 3 genes
  bla (?-lactamase), tnpA (transposase), and tnpB
  (resolvase, which functions in recombination)

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Examples of DNA-intermediate mobile elements

• Insertion Sequences (IS) elements in bacteria
• P elements in Drosophila
• AC/DS (dissociation) elements in maize
• AC is a full-length autonomous copy
• DS is a truncated copy of AC that is
  non-autonomous, requiring AC in order to
  transpose
• At least seven major classes of DNA transposons
  in the human genome (3 of total genome)

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Methods for Generation of Mutant Populations

• The most reliable method to ascertain gene
  function is to disrupt the gene and determine the
  phenotype change in the resulting mutant
  individual
• Two most popular methods to generate mutants
• 1. Insertional mutagenesis
• 2. Deletion mutagenesis

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Two main methods

• 1. Transposon insertion
• 2. T-DNA insertion

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Transposon mutagenesis

• Transposable elements or transposons
• sections of DNA (sequence elements)
• move, or transpose, from one site in the genome
  to another

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All transposable elements fall into one ofthe
following two classes

• 1. DNA elements
• 2. Retroelements

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DNA elements

• These elements transpose via DNA intermediates
  such as
• Ac/Ds and Spm in plants, P elements in animals,
  Tn in bacteria
• A common feature of DNA elements is the flanking
  of the element by short inverted repeat sequences
• The enzyme transposase recognizes these
  sequences, creates a stem/loop structure
• excises the loop from the region of the genome
• The excised loop can then be inserted into
• another region of the genome

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DNA-Immediate Mobile Genetic Elements

• The Short inverted repeats at the ends of the
  element
• These inverted repeats act as the substrates for
  recombination reactions mediated by the
  transposase

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Structure and transposition of a transposable
element
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Retroelements

• transpose via RNA intermediates
• The RNA is copied by reverse transcriptase into
  DNA
• the DNA integrates into the genome
• Retroelements are found in all eukaryotes
• such as Tos in rice, copia in animals and Ty1 in
  yeast

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Retrotransposon transposition
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Retorviruses

• The basic structure is an LTR long terminal
  repeat which flanks three genes,
• A complete retroviruses also contains three
  genes
• gag structural gene for capsid
• Pol reverse transcriptase
• env envelope gene for the virus

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How do we use a transposonfor mutagenesis?

• The insertion and excision of transposable
  elements
• result in changes to the DNA at the transposition
  site
• The transposition can be identified when a known
  DNA sequence or selection markers are inserted
  within the elements

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Transposomics

• EZTN Transposomes provide an efficient and
  reliable method for generating a library of
  random gene knockouts in vivo
• Gene inactivation and examination of the
  resulting phenotype will identify the function of
  the interrupted genes

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Transposon-Mediated Homologous RecombinationGene
Knockout in FungiHamer et al. 2001. Proc Natl
Acad Sci U S A. 2001 2498(9)5110-5
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T-DNA insertion mutagenesis

• T-DNA is a segment of the tumor-inducing (Ti)
  plasmid of Agrobacterium
• delimited by short imperfect repeat border
  sequences

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T-DNA transfer from Agrobacterium to plant cell
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Temperate bacteriophage Mu (Mu mutator)

• 37 kb linear DNA with central phage DNA and
  unequal lengths of host DNA at each end
• Mu integrates by transposition
• replicates when E. coli replicates
• During the lysogenic cycle, Mu remains integrated
  in E. coli chromosome

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bacteriophage Mu
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The advantages / disadvantage of Mu

• The advantages of the use of Mu are
• it is not normally found in the bacterial genome
• therefore there are few problems with homology to
  existing sequences in the chromosome in contrast
  to most other transposons
• Mu does not need a separate vector system
• since it is itself a vector
• A wide variety of useful mutants of Mu have been
  generated
• The disadvantage of Mu
• it is a bacteriophage and therefore can kill the
  host cell

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• Drosophila transposons
• 15 of Drosophila genome thought to be mobile
• 2 different classes
• Copia retrotransposons
• Conserved, 5-100 scattered copies/genome
• Structurally similar to yeast Ty elements
• Use RNA and reverse transcriptase
• Eye Color in Drosophila (white apricot wa)

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ITR(17bp)
ITR(17bp)
DTR
DTR
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P elements

• Hybrid dysgenesis, defects arise from crossing of
  specific Drosophila strains
• Occurs when haploid genome of male (P strain)
  possesses 40 P elements/genome
• P elements vary in length from 500-2,900 bp
• P elements code a repressor, which makes them
  stable in the P strain in male (but unstable when
  crossed to the wild type female/ female lacks
  repressor in cytoplasm)

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Ac (activator)/Ds (dissociation) System
discovered by B. McClintock (Noble Prize Winner
in 1983)
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Ac/Ds System
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Ac/Ds System
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Schematic Diagram of the Ds Donor Site
andPossible Transposition Events
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• Open arrowheads indicate the 5' and 3' ends of th
  transposon
• The Ds element carries the NPTII gene, which
  confers resistance to kanamycin (KanR)
• and a modified GUS reporter gene (Sundaresan
• et al. 1995 )
• Possible transposition events include the
  following
• (1) unlinked or loosely linked transposition to
  the same chromosome
• (2) transposition to a different chromosome
• (3) closely linked transposition and
• (4) closely linked transposition disrupting
  theIAAH gene

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Ac/Ds Transposon tagging system

• Advantages Efficient and cost-effective method
  to generate a large mutant population
• Disadvantages Secondary transposition
  complicates gene identification
• And transposon system is not available in many
  species

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• Transposition elements in Human

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Mobile Genetic Elements and Other Families of
Repetitive DNA

• The genome is littered with large families of
  repetitive sequences
• have no apparent function in the cell
• Mobile Genetic Elements
• Tandemly repeated simple sequence DNAs
• Satellite DNAs
• Short simple repeats (microsatellites)

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LINEs (Long interspersed elements)

• LINEs are one of the most ancient and successful
  inventions in eukaryotic genomes
• In humans, are about 6 kb long
• encode two open reading frames (ORFs)
• Most LINE-derived repeats are short, with an
  average size of 900 bp - 1,070 bp
• The LINE machinery is believed to be responsible
  for most reverse transcription in the genome

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SINEs (Short interspersed elements)

• short (about 100-400 bp)
• A single monophyletic family of SINEs (ALU)
• This family is the only active SINE in the human
  genome
• The human genome contains three distinct
  monophyletic families of SINEs the active Alu,
  and the inactive MIR and Ther2/MIR3

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Identification of a human specific Alu insertion
in the factor XIIIB gene

• Alu repeats are interspersed repetitive DNA
  elements specific to primates that are present in
  500,000 to 1 million copies
• An Alu Insert as the Cause of a Severe Form of
  Hemophilia A (factor VIII)
• Acta Haematol 2001106126129

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