Transposition mediated by RAG1 and RAG2
and its implications for the evolution of the
immune system
Light Chain
Heavy Chain
881609 Vicky Ting 2001.11.29
BACKGROUND

The genomic organization of the heavy- and lightchain gene segments

V(D)J recombination


Conserved heptamer and nonamer sequences flank the
gene segments encoding the V region of heavy(H) and
light(λ and κ) chains
12/23 rule

RAG1 and RAG2
recombination-activating genes
their products bind two recombination signals,
bring them into juxtaposition, and cleave the DNA,
thereby separating the signals from the flanking
coding segments.

HMG1 and HMG2 proteins
enhance the efficiency of coordinate cleavage.
Enzymatic
steps in the rearrangement of
immunoglobulin gene segments
How is transposition mediated
by RAG1 and RAG2?
1.An unexpected cleavage product

Material
32P-body-labelled
DNA fragments containing a 12 and 23signal orientated such that cleavage releases a signal
end/signal(SE/SE) fragment of roughly 320 bp,as well as
smaller fragments containing the hairpin coding ends.

Explore X:
cleavage using RAG
and HMG2 protein was
done for different
lengths of time at 37°C
and aliquots of the
reaction were analysed
on a native
polyacrylamide gel.
p.s * are bands resulting
from single cleavage at
the 12-or 23-signal.
 What is X?
by restriction mapping of purified band X DNA
indicated that it contain sequences from the
SE/SE fragment but not from the left or right
arms, and that it was not the result of end-to-end
ligation of the SE/SE fragment.
Find the structure of the band X

Material
Bal31, which is a doubled stranded DNA exonuclease and
a single stranded DNA exo- and endonuclease.
RAG
HMG2
32P-body-labelled DNA fragments

Footprinting
In the presence of
RAG and HMG2
proteins, band X and
the SE/SE fragment
were substantially
more resistant to
Bal31 than were
other DNA fragment.
indicate X might be
stable associated
with the RAG and
HMG2 proteins

Immunoprecipitation
also prove that band X
is stable bound by
RAG1, RAG2 and
HMG2.
ps. α myc
RAG1 and RAG2
contain the epitope tag.


Whether band X could be generated using
the SE/SE fragment as the starting substrate?
Material
SE/SE fragment DNA
PCR
incubation with RAG, GST-RAG and HMG proteins


band X digested with
PstI (restriction enzyme)
The reduced mobility
of PstI-digested band X
relative to the SE/SE
fragment may be due to
single stranded gaps in
the band X DNA

Material
an 32P-body-labelled
SE/SE fragment
purified from a
cleavage reaction.
band X requires a
substrate with
two signal ends
2.intramolecular transposition

The reason X(Pstl) is
longer than SE/SE
fragment is that X(Pstl)
is not merely the end-toend ligation of SE/SE
fragment, but maybe
due to intramolecular
transposition.
 Same strand attack
 Opposite strand attack

When gap is filled in, target-site duplication is formed.
So that A is longer than B in Fig.3a .

Structure of the two strands of the inversion circles

Material
band X was generated from SE/SE fragment labelled at the
5’ end of either the 12-or the 23- signal and gel purified.
8% denaturing polyacrylamide/urea gel
restriction enzyme that cut close to the end.
PstI and CalI
one strand must contain two nicks.
Result

The RAG proteins constitute a transposase.
Together with HMG2,they can proform
intramolecular transposition.
3.intermolecular transposition


Southern blotting
Material
1.4 kb SE/SE fragment
containing a tetracyclineresistance gene and a 6.3
kb circular target plasmid
containing a ampicillinresistance gene.
RAG and HMG2 proteins.

Formation of this product
required the target
plasmid and the RAG and
HMG2 proteins.
results show that
RAG and HMG2
proteins can mediated
intermolecular
transposition into
diverse target
sequence.
Discussion
RAG-mediated
transposition model
The importance of HMG2
Evolution
RAG transposon
Bony fish
Jawed
vertebrates
Cartilaginous fish
vertebrates
Jawless
vertebrates

Evolution of the vertebrate
immune system
Thanks for your listening!
Make a wish!
4b.
4c.
6c.