Genome Instability and Repair

1
Plant Variegation (or sectoring) - often genetic
but can be other causes
Kiwi vine
Dianthus
Begonia
Polemonium
Plants Delight Nursery
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Variegation is usually nuclear- determined but
sometimes cytoplasmically inherited -in this
case, Mirabilis (4-oclock) via the chloroplast
Baur, Correns
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Genome Instability and Repair

• Genome Instability Transposable Elements
• 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 called them "controlling elements because of
  the myriad effects on gene expression.

4
1. Nobelprize.org 1983 Nobel Prize in
Physiology and Medicine - her first paper on
this was published in 1948 2.
profiles.nlm.nih.gov/LL/
Barbara McClintock 1902-1992
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Other characteristics of McClintock's
"controlling elements"

• 1. Elevate the mutation rate.
• 2. Cause unstable mutations that often revert
  partially, sometimes giving new phenotypes.
• 3. Often move during meiosis and mitosis.
• 4. Movement (and resulting mutations) are
  accelerated by genome damage.

6
Some maize phenotypes caused by transposable
elements excising in somatic tissues. Parental
plants are mutants defective in starch synthesis
(endosperm phenotypes) or anthocyanin synthesis
(aleurone and pericarp phenotypes).
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Molecular Analysis of Transposons

• Transposable elements (or Transposons) were first
  cloned by cloning a gene from wild-type plants
  that they often inactivated (Federoff lab).
• The cloned DNA was used to isolate the gene from
  mutant lines. This process is also called
  "Transposon trapping.

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Common features

• Exist as multiple copies dispersed in the
  genome.
• Insertion site of element does not have extensive
  homology to the transposon.
• Contain inverted repeats at element termini.
• A short, direct repeat of genomic DNA often
  flanks the transposon (i.e., integration results
  in a short duplication of target sequence).
• Autonomous elements encode proteins that
  mobilize the element.

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(No Transcript)
10
Features unique to plant transposons

• Footprints when some elements move, leave
  behind duplicated target sequence (footprint),
  which can still affect the gene (only partial
  restoration of gene function).
• Two-element systems mobility of one element
  depends on another.

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How duplications in the target site probably
occur.
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Molecular Bases of the Myriad Effects of
Transposons on Gene Expression

• Insertions don't necessarily inactivate genes,
  effects can be complex
• Insertion into a promoter can alter
  tissue-specific expression.
• Many elements have their own promoters.
• With insertion into an exon, the element can
  sometimes be completely spliced out at the RNA
  level.
• Or the inserted transposon can donate new splice
  sites generating new protein variants.

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Ac/Ds elements

• Described genetically by McClintock in maize
• Ds - dissociation locus (chromosomal breaks),
  semi-autonomous element, its mobility depends on
  Ac
• Ac - Activator, autonomous element
• Cloned from the waxy (Wx) locus, which encodes
  UDPglucose-starch transferase

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• Ds derived from Ac, contains internal deletions.
• Both elements contain an 11-bp inverted repeat
  at the termini (TIR)
• Subterminal regions also contain repeated
  sequences.
• Both subterminal and TIRs needed for
  transposition, recognized by the Transposase.

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Structure of Ac and its Transposase
Kunse Weil, 2002
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En/Spm family of Transposons

• En/Spm are autonomous elements and are
  essentially identical.
• also first cloned from Waxy locus
• contain 13-bp TIR at ends
• Also contain subterminal repeats
• Some preference for inserting into DNA with
  homology to subterminal repeats.

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• Spm is 8.5 kb and has 2 main ORFs
• Alternative splicing produces 4 major
  transcripts and proteins (tnpA-D).
• tnpA binds subterminal repeats.
• tnpD binds the TIR and is probably the
  endonuclease.
• Also a 2-element system dSpm is defective
  version, contains internal deletions, and
  movement depends on Spm.

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Structure of the En/Spm Element
Kunse Weil 2002
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Proposed Mechanism of Spm transposition
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Mu/MuDR (Mutator)

• Discovered in maize differs significantly from
  Ac and En/Spm families
• Many copies per nucleus (autonomous and
  non-autonomous versions)
• Contains a long TIR (200 bp)
• Transposes via a gain/loss (somatic cells) or a
  replicative (germline cells) mechanism.

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Structure of MuDR (autonomous Mu) and its
promoters.

• MuDrA and B expressed at high levels in dividing
  cells and pollen, because of transcriptional
  enhancers.
• MURA (mudrA) is transposase has NLS.
• MURB needed for insertion in somatic cells.

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Mu elements moving to new sites in a cross
between a Mu-active strain (or line) and a maize
line lacking Mu.
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• Retrotransposons
• - similar to retroviruses
• - move by RNA intermediate
• -encode a reverse transcriptase activity
• can be many thousands of copies in the genome

Fig. 7.34 in Buchanan et al.
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Retro-transposons in pea (Pisum sativum) genome
Macas et al. (2007) BMC Genomics 8427
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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.
• Methylation can also block binding of the
  Transposase (and other trans-factors) to the
  subterminal and TIR

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Biological Significance of Transposons

• They provide a means for genomic change and
  variation, particularly in response to stress
  (McClintocks "stress" hypothesis) (1983 Nobel
  lecture, Science 226792)
• or just "selfish DNA"? Or both?
• No known examples of an element playing a normal
  role in development.

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Using transposons to isolate genes - "Transposon
tagging"

• Can be extremely powerful, isolate gene based on
  an interesting mutant phenotype, for example, a
  regulatory gene.
• Strategy
• Identify mutant caused by transposon insertion
  (i.e., demonstrate tight genetic linkage between
  mutant phenotype and presence of a copy of the
  transposon).
• Fish out the gene with the inserted element from
  a genomic library of mutant DNA (use cloned
  transposon as probe).
• Use mutant gene to fish out the wild-type gene.

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• Possible limitations
• 1. Must use organism with known active elements.
• - If there are no characterized elements, use
  heterologous ones introduced by
  transformation
• 2. Element must integrate into the desired gene.