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) , its 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
Barbara McClintock 1902-1992
1947 at Cold Spring Harbor

• Nobelprize.org
• 1983 Nobel Prize in Physiol. Med.
• - her first paper on this topic was published
  in 1948
• 2. profiles.nlm.nih.gov/LL/

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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 seed
tissues. Parental plants are mutants defective
in starch (endosperm phenotypes) or anthocyanin
(aleurone and pericarp phenotypes) synthesis.
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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

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

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(No Transcript)
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Features unique to plant transposons

1. Footprints when some elements move, leave
   behind duplicated target sequence (footprint),
   which can still affect the gene (only partial
   restoration of gene function).
2. 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.
• Most elements have their own promoters.
• With insertions in an exon, elements are
  sometimes 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 (caused 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 is 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.

15
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
20
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., MurA of Mu (TpnA of Spm)
• Transcriptional silencing mechanism not well
  understood, but correlates with methylation of
  the promoter (similar to heterochromatin).
• 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)
• e.g., LINE retrotransposons in humans can/have
  caused
• Local genome instability
• Genomic rearrangements, new exons, etc.
• (Cordaux Batzer (2010) Nat. Rev. Genet. 10,
  691-703)
• 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.