Transposable Genetic Elements

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Transposable Genetic Elements
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Babara McClintock
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Transposable Genetic Elements
Contents

• Controlling Elements in Maize
• Bacterial Insertion Sequences
• Prokaryotic Transposons
• Mechanism of Transposition in Prokaryotes
• Review of Transposable Elements in Prokaryotes
• Molecular Nature of Transposable Elements in
  Eukaryotes
• Review of transposable Elements in Eukaryotes

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Transposable Elements
o  Segments of the genome that are capable of
moving around to different locations -
Transposable elements usually are flanked by
repeated sequences - often carry transposase
genes that confer the transposition ability  
Two basic types of transposition
o  Conservative transposition An element
leaves one locations and inserts in another
Does not change overall copy number o  
Replicative transposition A copy of a
transposable element is made and this inserts in
a new place This is the most common form
of transposition
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Controlling elements in maize

• McClintocks experiments the Ds element
• The wx (waxy) locus
• General characteristics of controlling elements

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Controlling elements in maize

• 1938 Marcus Rhoades reported an odd finding in
  phenotypic ratios of corn...
• Self pollination of a true-breeding pigmented
  corn kernel yielded 12 3 1 ratio
• of pigmented dotted unpigmented kernels
  dominant epistasis

• He first hypothesized that two events had
  occurred at unlinked loci
• 1. Pigment gene A1 had mutated to colorless
  mutant a1
• 2. At another locus, a dominant allele for
  dotting (Dt) had appeared

• What was causing the dotted phenotype?
• - Reverse mutation of a ? A
• Pollen from a/aDt/- plants X a/a tester
• ? some are completely pigmented type
• a unstable mutant allele (high reversion rate)
• the allelic instability is dependent on the
  unlinked
• Dt gene

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McClintocks experiments the Ds element

• 1950's McClintock reported the presence of a
  genetic factor Ds (for "Dissociation")
• whose presence caused a high degree of
  chromosome breakage wherever it appeared
• The action of Ds is another type of instability
• The instability of Ds turned out to depend on
  the presence of an unlinked gene
• Ac (for "Activator")
• Ac Ds locus was constantly changing position

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The transposition of Ds into the C gene in corn

• CDs/CDsAc/Ac x cDs/cDsAc/Ac
• C means pigment expressed
• Ds and Ac indicate the lack of the element
• Cu indicates the insertion of Ds into the C
  locus, which converts
• it to UNSTABLE colorless!
• aDt system ? CuAc system
• some specificity prevents this
  cross-activation of
• mutational instability (aAc or CuDt)

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• General characteristics of controlling elements

Target gene / receptor element / regulator gene
controlling
elements Nonautonomous unstable allele revert
only in the presence of the regulator Autonomous
unstable allele - Ds is an incomplete version of
Ac itself
Summary of the main effects of controlling
elements in corn
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Mosaicism through transposon mutagenesis
snapdragon
corn
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Bacterial insertion sequence

• segments of bacterial DNA that can move from one
  
• position to a different position

Physical demonstration of DNA insertion
- IS (insertion sequences) were first discovered
in the gal operon of E. coli - Mutation by
insertion is demonstrated with phage lamda
particle carrying the bacterial gene for
galactose utilization (gal) or the mutant gene
gal- - The increased density of gal- particles
was caused by the insertion of DNA
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Bacterial insertion sequence
Direct visualization of inserted DNA
The single stranded loop is caused by the
presence of an insertion sequence in ?dgalm
Electron micrograph of a ?dgal/ ?dgalm DNA
heteroduplex
Each heteroduplex shows a single stranded buckle,
or loop
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Bacterial insertion sequence
Identification of discrete IS elements

• Are the segments of DNA that insert
• into genes merely random DNA fragments
• or are they distinct genetic entities?
• Hybridization experiments
• Cross-hybridization
• Distinct elements
• The genome of the standard WT E.coli
• is rich in IS elements
• - these elements are mobile

Insertion sequences are also commonly observed in
the F factor
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Bacterial insertion sequence
Orientation of IS element
Heteroduplex DNA structure
The hybrid can be explained by assuming that the
IS1 sequence is inserted in opposite directions
in the two mutants
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Prokaryotic transposons

• Physical structure of transposons
• Movement of transposons
• Phage mu

R factors
Mode of action of plasmids
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Prokaryotic transposons
Physical structure of transposons

• Peculiar structure formed when denatured plasmid
  DNA is reannealed
• Double stranded IR region separates a large
  circular loop from the
• Small lollipop loop

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Prokaryotic transposons
Physical structure of transposons

• The IR sequences are a pair of IS elements in
  many cases
• The IR sequences together with their contained
  genes
• ? transposon (Tn)

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Prokaryotic transposons
Physical structure of transposons
The insertion of a Tn into a plasmid RTF
resistance-transfer functions
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Prokaryotic transposons
Movement of transposons
Each transposon can be transferred independently
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Prokaryotic transposons
Phage mu

• a normal-appearing phage
• 36,000 nucleotide long
• can insert itself anywhere in a bacterial or
  plasmid genome
• in either orientation mutation in the genome
  like IS
• each mature phage particle has on each end a
  piece of flanking DNA
• from its previous host ? no insert into genome
  in next generation
• contain its own IR sequences but not in the
  chromosome ends
• genetic snap fastener phage mu can mediate the
  insertion of phage ?
• or drug resistant gene into a bacterial
  chromosome using 2 mu genome

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Prokaryotic transposons
Phage mu
Phage mu can mediate the transposition of a
bacterial gene into a plasmid Phage mu can cause
deletion or inversion of adjacent bacterial
segment
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Mechanism of transposition in prokaryotes
Replicative transposition
A new copy of the transposable element is
generated in the transposition event The
structure of transposon 3 Transposition of Tn3
takes place through a cointegrate intermediate
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Mechanism of transposition in prokaryotes
Conservative transposition

• Some transposons excise from the chromosome and
  integrate
• Into the target DNA
• Generation of heteroduplex and
• homoduplex Tn10 elements

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Mechanism of transposition in prokaryotes
Conservative transposition

• Consequences of conservative
• Replicative transposition
• Tn10 transpose by excising
• themselves from the donor DNA
• integrating directly into the
• recipient DNA

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Mechanism of transposition in prokaryotes
Molecular consequences of transposition

• Duplication of a short sequence of nucleotides
• in the recipient DNA is associated with the
  insertion of a
• Transposable element
• The number of base pairs is
• a characteristic of each
• element

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Mechanism of transposition in prokaryotes
Rearrangements mediated by transposable elements

• Transposons generate a high incidence of
  deletions in their vicinity

• When varying lengths of the surrounding DNA are
  excised
• along with the transposon IMPRECISE
  EXCISION
• When the transposon is excised and the gene that
  was disrupted by the insertion
• are restored PRECISE EXCISION
• (This occurs rarely, compared to imprecise
  excision)

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Review of transposable elements in prokaryotes
1. There are several different types of
transposable elements IS, transposons and
phage µ 2. Two copies of a transposable
element can act in concert to transpose the
DNA segments in between them (the transposon
containing antibiotic resistance gene) 3. Most
transposable elements have recognizable inverted
repeat structures 4. Transposable elements are
found in plasmids, and in bacterial chromosomes
5. Transposable elements usually generate repeats
after insertion 6. Detailed mechanism of
transposition isn't yet known!
(replicative conservative)
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Molecular nature of transposable elements in
eukaryotes

• Transposable elements are even more prevalent in
• eukaryotic chromosomes than in bacterial
  chromosomes
• 1. Retroviruses ss RNA animal virus

The life cycle of a retrovirus
Transposition by retrovirus
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Molecular nature of transposable elements in
eukaryotes

• 2. Retrotransposons
• - transposable elements that
• utilize reverse transcriptase to
• transpose through an RNA
• intermediate
• - two types
• viral nonviral
• Viral retrotransposon
• similar to retrovirus
• - Yeast Ty elements
• 35 copies in yeast genome
• generate a repeated sequence of target DNA
  during transposition

Schematic representation of a viral
retrotransposon
The structure of a yeast transposable element
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Molecular nature of transposable elements in
eukaryotes
The mechanism for transposition of
retrotransposon

• The addition of galactose greatly increases
• the frequency of transposition of the
• altered Ty element
• - The transpose Ty DNA contains no intron
• Transposition occurred through
• RNA intermediate

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Molecular nature of transposable elements in
eukaryotes
- copia-like elements in Drosophila LTR
at least, seven families generate a repeated
sequence of target DNA during
transposition - A comparison of the genes of
integrated retrovirus DNA and the yeast Ty
elements and Drosophila copia elements
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Molecular nature of transposable elements in
eukaryotes
Nonviral retrotransposons LINES and SINES -
Frequent in mammals - Some sequence divergence
Repetitive elements found in the human gene (HGO)
encoding Homogentisate 1,2-dioxygenase, the
enzyme whose deficiency cause alkaptonuria
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Molecular nature of transposable elements in
eukaryotes
Function of transposable elements - Mutation of
genes by insertion - Regulation of gene
expression by insertion - Transposable
element-mediated rearrangements deletion,
duplication, and inversion ? new genome, new
function during evolution
Uses of transposable elements - prokaryotes
conventional antibiotic-resistance marker -
eukaryotes generation of insertion mutations,
mapping, gene cloning,
transgenic organism
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Molecular nature of transposable elements in
eukaryotes

• P elements in Drosophila
• - 2.9kb with 31 base inverted repeats
• encodes a transposase
• - Do not utilize an RNA intermediate during
  transposition
• - Can insert at many different positions
• - Transposition is controlled by
• repressors encoded by the element
• - P elements with internal deletions
• can be mobilized and then remain
• fixed in the new position
• transposon tagging

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Using P elements to insert genes
P-element mediated gene transfer in Fly.
- P elements mobilize only in germ-line cells
ry-/ry-
Phenotype observation
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Review of transposable elements in eukaryotes

• Transposable elements exist in all cells
• (yeast, drosophila, maize, and mammals)
• Some elements can be used as tools for cloning
  and gene manipulation
• (P element insertion of genes into germ lines
  of recipient cells)
• Short repeated sequences are generated at the
  site of insertion in the
• target site
• Some eukaryotic transposable elements use an RNA
  intermediate during
• insertion this is not seen in prokaryotes