Download Gene Tagging with Transposons

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.
(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!