Transposition%20mediated%20by%20RAG1%20and%20RAG2%20and%20its%20implications%20for%20the%20evolution%20of%20the%20immune%20system

1
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
2
BACKGROUND

• The genomic organization of the heavy- and
  light-chain gene segments

3

• V(D)J recombination

4

• Conserved heptamer and nonamer sequences flank
  the gene segments encoding the V region of
  heavy(H) and light(? and ?) chains

• 12/23 rule

5

• 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.

6

• Enzymatic steps in the rearrangement of
  immunoglobulin gene segments

7
(No Transcript)
8
How is transposition mediated by RAG1 and
RAG2?
9
1.An unexpected cleavage product

• Material
• 32P-body-labelled DNA fragments containing a
  12 and 23-signal 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.

10

• Explore X
• cleavage using RAG and HMG2 protein was
  done for different lengths of time at 37C 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.

11
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.

12
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

13

• 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

14

• Immunoprecipitation
• also prove that band X is stable bound by
  RAG1, RAG2 and HMG2.
• ps. a myc
• RAG1 and RAG2 contain the epitope tag.

15

• 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

16

• 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

17

• Material
• an 32P-body-labelled SE/SE fragment purified
  from a cleavage reaction.
• band X requires a substrate with two signal
  ends

18
2.intramolecular transposition

• The reason X(Pstl) is longer than SE/SE fragment
  is that X(Pstl) is not merely the end-to-end
  ligation of SE/SE fragment, but maybe due to
  intramolecular transposition.

19

• Same strand attack

20

• Opposite strand attack

• When gap is filled in, target-site duplication is
  formed.
• So that A is longer than B in Fig.3a .

21

• 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.

22
(No Transcript)
23
(No Transcript)
24
Result

• The RAG proteins constitute a transposase.
  Together with HMG2,they can proform
  intramolecular transposition.

25
3.intermolecular transposition

• Southern blotting
• Material
• 1.4 kb SE/SE fragment containing a
  tetracycline-resistance gene and a 6.3 kb
  circular target plasmid containing a
  ampicillin-resistance gene.
• RAG and HMG2 proteins.

26

• 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.

27
Discussion

• RAG-mediated transposition model
• The importance of HMG2

28
Evolution
RAG transposon
Bony fish
Jawed vertebrates
Cartilaginous fish
vertebrates
Jawless vertebrates
29

• Evolution of the vertebrate immune system

30
Thanks for your listening!
Make a wish!
31
4b.
32
4c.
33
6c.