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Genome Instability and Repair

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Begonia. Dianthus. Polemonium. Plants Delight Nursery. Kiwi vine. Plant Variegation (or sectoring) ... DNA elements capable of moving ('transposing') about the ... – PowerPoint PPT presentation

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Title: Genome Instability and Repair


1
Plant Variegation (or sectoring) - often genetic
but can be other causes
Kiwi vine
Dianthus
Begonia
Polemonium
Plants Delight Nursery
2
Variegation is usually nuclear- determined but
sometimes cytoplasmically inherited -in this
case, Mirabilis (4-oclock) via the chloroplast
Baur, Correns
3
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
5
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).
7
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.

8
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.

9
(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.

11
How duplications in the target site probably
occur.
12
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.

13
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

14
  • 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.

15
Structure of Ac and its Transposase
Kunse Weil, 2002
16
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.

17
  • 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.

18
Structure of the En/Spm Element
Kunse Weil 2002
19
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.

21
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.

22
Mu elements moving to new sites in a cross
between a Mu-active strain (or line) and a maize
line lacking Mu.
23
  • 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.
24
Retro-transposons in pea (Pisum sativum) genome
Macas et al. (2007) BMC Genomics 8427
25
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

26
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.

27
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.

28
  • 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.
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