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Genome Instability and Repair

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MuDrA and B expressed at ... Arial Times New Roman Calibri Default Design Slide 1 Slide 2 Genome Instability and Repair Slide 4 Other characteristics of ... – 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) , its 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
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/

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

9
(No Transcript)
10
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.

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.
  • 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.

13
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

14
  • 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
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., 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

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)
  • 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.

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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