Transcript Gene Tagging with Transposons

Transposable
Genetic Elements
MBIOS 520/420
September 22, 2005
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Transposon Introduction
• Transposable elements are stretches of DNA that can move
to new locations in a genome
• These elements can contain genes or be non-coding
• Large portions of higher eukaryotes’ genomes are composed
of either inert or active transposons (often as repetitive DNA)
• Transposons are thus important evolutionarily
• Transposons can also be used to isolate genes or introduce
foreign genes into cells
Bacterial Transposon Discovery
• First transposons characterized, these are the simplest
• Detected because experimental lac- strains kept reverting
back to wild type (ie, colonies kept turning blue)
• lac- mutants were due to transposons which then moved back
out of the gene
Agar w/X-Gal
transposon
Agar w/X-Gal
Transposition
event
lac gene
lac gene
lac gene
Bacterial Transposons
• Can occur in bacterial genome or in plasmid, and can move
between these two
• Consist of two major types:
Insertion Sequences (IS)

small, <2500 bp
Composite Transposons

large, flanked by
two IS elements
Insertion Sequences
• Consists of a pair of inverted terminal repeats at each end
(cannot be mutated without loss of transposition activity)
• Between this is a stretch of DNA, often containing the gene
for transposase – the enzyme that catalyzes transposition
• Flanking the terminal repeats are a pair of direct repeats that
result from the transposition process
Insertion Sequence Transposition
Transposase moves the
element by creating a
staggered cut at either end in a
random spot of the genome
The IS element then
moves then inserts into
this region
DNA polymerase fills in
the resulting singlestranded areas
The result is termed a
target site duplication
Composite Transposons
• Denoted Tn
• Created when two IS elements insert
near each other
• The elements can be either in inverse or
direct orientation to each other
• These two then move together and
transpose the sequence between them
(often carrying genes)
• Movement of these large elements is
how bacteria become antibiotic
resistance (often using viral
intermediates)
Composition Transposon Transposition
• Involves both IS elements
• Two types:
Replicative Transposon

transposon is copied & moved
(ie, a copy remains in place)
Non-Replicative Tranposon
(aka “cut & paste”)

the whole element is moved
(no copy remains)
• Similar to conserved & semi-conservative DNA replication
Tn3 Transposition
• A combination of replication & recombination
Tn10 Transposition
How can we determine if a transposon uses replicative or “cut and paste” transposition?
Gene Tagging with Transposons
• We can use transposons to tag and isolate genes
• Ex: let’s say we want to isolate a blue flower color gene
STEP 1
transposon
A. Transform plant with a
vector containing a
transposon.
M2
(Thousands of plants needed,
depending on genome size)
transposon
Blue color gene
B. Grow progeny from that
plant and pick out the mutant
phenotype (in this case, an
uncolored flower).
C. This plant should have the
transposon inserted
somewhere in the gene.
Gene Tagging with Transposons
STEP 2
A. Isolate genomic DNA from
the mutant plant.
BAC clones
(many thousands of these)
B. Make genomic DNA library
from this sample (ex: using
BAC vectors).
C. Pool clones of the library
into 96-well plate.
THE MATH
Ex: Rice
Genome size = 400 million bases
BAC insert size = 200,000 bases
# clones needed for 1X = 2,000
# clones needed for 6X = 12,000
# of clones per well = 125
Gene Tagging with Transposons
PCR w/ transposon primers
1
2
3
4
5
STEP 3
1
2
3
4
5
A. Find the well containing the
BAC clone with the
transposon (use PCR).
B. Grow the cells from this well
on a plate.
P32-labeled
transposon
probe
C. Hybridize with transposon
probe to locate exact colony
that has the clone.
D. Sequence this clone.
You’ve found the gene!
Gene Tagging with Transposons & Inverse PCR
• There is a faster way of identifying the gene without having to
build an entire library, by using a technique called inverse PCR
STEP 2b
EcoRI
A. Follow STEP 1 like before.
Isolate mutant & extract its
DNA.
B. Cut DNA with restriction
enzymes, then re-ligate to
form circular segments.
DNA ligase
(high volume
reaction)
SEQUENCE
C. Use the transposon-based
primers & do PCR. This
amplifies the flanking gene
regions. Sequence it.
Gene Tagging with Transposons - Troubleshooting
• What if we get many transposons inserting into our mutant
genome? How do we tell which one is in our color gene?
OPTION 1
Do inverse PCR as in STEP 2B.
SEQUENCE
BLAST sequence & search for
similarity with other known
pigment genes.
(GENBANK)
Gene Tagging with Transposons - Troubleshooting
OPTION 2
Cross mutant plant back to
wild-type plant.
Produce an F2 (or more)
generation.
Do Southern blot with
tranposon probe. Find marker
that segregates with mutation.
Many plants needed.
P
X
F1
F2
λ HindIII
Marker
23.0 kb
9.4 kb
6.5 kb
4.3 kb
2.3 kb
2.0 kb
Make a library out of a plant
with only this marker (Plant D
in our example).
0.5 kb
A
B
C
D E F
Gene Tagging with Transposons - Troubleshooting
OPTION 3
A
B
C
D
Pick out all BAC clones with
transposons via PCR (STEP 3).
Do RFLP ofF2 mutant plant
using transposon as a probe.
λ HindIII F2 Mutant
Marker
Plant
23.0 kb
Cut BACs with same restriction
enzyme as in your RFLP. Hybe
with tranposon probe.
Transposon probe should bind
at same MW in mutant and
BAC digest.
9.4 kb
6.5 kb
4.3 kb
2.3 kb
2.0 kb
0.5 kb
A
B
C
D
Eukaryotic Transposons
• Similar in structure to bacterial transposons
• Most are thought to be derived from viral genomes that have
integrated into a host cell genome
• Some eukaryotic transposons move via an RNA intermediate
• Some transpositions are utilized for programmed genome
rearrangements
• Movement of transposons in genomes can inactive or activate
genes, and can cause cancer
• The movement and buildup of transposable sequences has
had an effect on the evolution of eukaryotice genomes
Maize Transposons
• Two best characterized transposons are Ac (Activator) and Ds
(Dissociation) elements (both are “cut & paste” types)
• Can occur in many copies of the cell, but must work together
• Ac is ~4.6 kb, with same basic
structure as insertion elements
• Ds is similar (identical inverted
terminal repeats) but has
deletions of various sizes in it
Transposase Gene
Transposase Missing
• Because of the deletions, Ds
does not have transposase &
cannot transpose itself (Ac
needed)
Discovery of Transposons
• Barbara McClintock,
maize geneticist
• Observed mosaic corn
kernels, despite presence of
CI, a dominant colorless allele
• Concluded that chromosome
was breaking, causes CI loss
• Only occurred when another
segregating factor, Ac, was
present
CI Present
CI Absent
Using Ac & Ds Elements for Mutagenesis
• When we do transposon
mutagenesis, how can we
control when transposition
stops & starts?
• Ac can be introduced via a
vector
Ac+/+ Ds-/P
Ac-/- Ds+/+
X
Ac+/- Ds+/-
F1
F2
• But how do we know that
Ac won’t transpose into our
gene instead of Ds?
Ac-/-
Ac+/? Ds+
X
Mutate the inverted
terminal repeats of Ac,
then it can’t transpose!
BC1F1
BC1F2
Stable
Mutant
Ac-/- Ds+
Drosophila Transposons
• Known as P elements, similar in structure to Ac & Ds
• P elements can be incomplete (no transposase) or complete
(functional transposase) – analogous to Ac/Ds
• P elements are only active in
germ line cells, because a stop
codon exists in transposase
• In germ cells, alternative splicing
removes exon 2 to remove the
codon
• Demonstrated by engineering a P
element without intron 3
Retro-Transpososons
• These transpose via an RNA intermediate
• Transposon is transcribed into RNA, then reverse
transcriptase creates “cDNA” that inserts into genome
• Two categories exist, based on their origin & structure:
Retroviruslike Elements 
Retroposons

Possess Long Terminal Repeats
Have viral genes gag & pol
Derived from retroviruses
Non-LTR retrotransposons
Have poly A:T tract
Derived from reverse transcribed mRNA
Retroviruslike Elements
• Ty1 of yeast is best studied
• Long terminal repeats called δ
regions (not inverted) flank a
coding region
• Coding region has TyA & TyB;
these genes are gag & pol derived
• ~35 copies per yeast cell;
sometimes solo δ regions are
found (formed by recombination
between δ)
• Form target site duplications
Ty1 Transposition
• RNA is synthesized by normal
RNA pol II transcription
• δ elements act as strong
promoters (can activate genes)
• TyB gene has reverse
transcripase activity and
produces dsDNA from the RNA
• DNA integrates into the genome
(δ element is replicated)
• copia and gypsy are Drosophila
retrotransposons very similar to
Ty1 (gypsy even has viral env
gene)
Proof of Ty1 Transposition
• How can we prove Ty1 transposes as an RNA molecule?
• Constructed Ty1 element with a galactose-inducible promoter
and an intron
• Used galactose to stimulate transcription, then found that all
the new copies transposed had the intron spliced out
Retroposons
• F, G and I elements in Drosophila; LINEs in humans
• Also called non-LTR retrotransposons because they lack
inverted or direct repeats at their ends (do have target site
repeats)
• Retroposons all have a poly-A region at the end, evidence that
these are reverse transcribed mRNAs that re-inserted in the
genome
• These function by reverse transcription, followed by insertion
• LINE-1 or L1 = only known active human transposon (make up
5% of human genome) & can cause mutations (ex: hemophilia)
Transposition & Chromosome Rearrangement
• Can create duplications,
deletions, inversions,
translocations
• Means of creating
pseudogenes – no
selection pressure (can
gain novel function)
Deletions & Duplication Created by
Transposition:
Transposition & Chromosome Rearrangement
Deletions & Inversions
Created by
Transposition:
Transposition & Cancer
• Antibody genes have powerful enhancers and use
recombination to produce diverse sets of antibodies
• c-myc can recombine with this region & cause cancer
Enhancer Trapping
• Technique use transposons
to identify tissue-specific
enhancers
• Add lacZ gene between
the inverted terminal repeats
of a P element
• Transform Drosophila
with P element
• Dissect Drosophila & grind
tissue in X-Gal
• Blue color change shows
that P element inserted near
a tissue specific enhancer
Or do
inverse PCR
Transposons & Genome Evolution
• All active transposons have the potential to cause mutations
• These can be deleterious or potentially beneficial
• Duplication of genes via chromosome rearrangements can
produce pseudogenes which can eventually gain new function
• Which came first  Retroviruses or Retrotransposons?