Title: Genome Instability and Repair
1Plant Variegation (or sectoring) - often genetic
but can be other causes
Kiwi vine
Dianthus
Begonia
Polemonium
Plants Delight Nursery
2Variegation is usually nuclear- determined but
sometimes cytoplasmically inherited -in this
case, Mirabilis (4-oclock) , its via the
chloroplast
Baur, Correns
3Genome 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.
4Barbara 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/
5Other 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.
6Some 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.
7Molecular 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.
8Common 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)
10Features 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.
11How duplications in the target site probably
occur.
12Molecular 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.
13Ac/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.
15Structure of Ac and its Transposase
Kunse Weil, 2002
16En/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.
18Structure of the En/Spm Element
Kunse Weil 2002
19Proposed Mechanism of Spm transposition
20Mu/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.
21Structure 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.
22Mu 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.
24Retro-transposons in pea (Pisum sativum) genome
Macas et al. (2007) BMC Genomics 8427
25Control 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
26Biological 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.
27Using 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.