Download Genome Instability and Repair

Plant Variegation (or
sectoring)
Kiwi
vine
- often genetic but can be other
causes
Begonia
Dianthus
Polemonium
Plants Delight Nursery
Variegation is
usually nucleardetermined but
sometimes
cytoplasmically
inherited
-in this case,
Mirabilis (4o’clock) , its via
the chloroplast
Baur, Correns
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.
Barbara McClintock
1902-1992
1947 at Cold Spring Harbor
1. Nobelprize.org
1983 Nobel Prize in Physiol. & Med.
- her first paper on this topic was
published in 1948
2. profiles.nlm.nih.gov/LL/
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.
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.
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“.
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.
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.
How duplications
in the target site
probably occur.
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.
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
•
•
•
•
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.
Structure of Ac and its Transposase
Kunse & Weil, 2002
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.
• 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.
Structure of the En/Spm Element
Kunse & Weil 2002
Proposed Mechanism of Spm transposition
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.
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.
Mu elements
moving to new
sites in a
cross between
a Mu-active
strain (or line)
and a maize
line lacking
Mu.
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.
Retrotransposons
in pea
(Pisum
sativum)
genome
Macas et al. (2007) BMC Genomics 8:427
Control of Transposons
1.
Autoregulation: Some transposases are transcriptional
repressors of their own promoter(s)
• e.g., MurA of Mu (TpnA of Spm)
2.
Transcriptional silencing: mechanism not well
understood, but correlates with methylation of the
promoter (similar to heterochromatin).
3.
Methylation can also block binding of the Transposase
(and other trans-factors) to the subterminal and TIR
Biological Significance of Transposons
• They provide a means for genomic change and variation,
particularly in response to stress (McClintock’s "stress"
hypothesis: 1983 Nobel lecture, Science 226:792)
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.
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:
1. Identify mutant caused by transposon insertion
(i.e., demonstrate tight genetic linkage between
mutant phenotype and presence of a copy of the
transposon).
2. Fish out the gene with the inserted element from
a genomic library of mutant DNA (use cloned
transposon as probe).
3. Use mutant gene to fish out the wild-type gene.
•
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.