Generating a Repertoire of Antigen-Specific 
Receptors 
W9 
David G. Schatz, Ph.D. — Assistant Investigator 
Dr. Schatz is also Assistant Professor of Immunobiology at Yale University School of Medicine. He received 
undergraduate degrees in molecular biophysics and biochemistry from Yale University and in philosophy 
and politics from Oxford University. His Ph.D. degree and postdoctoral training were done with David 
Baltimore at the Massachusetts Institute of Technology and the Whitehead Institute. 
CELLS of the immune system act in concert to 
protect against infectious agents and trans- 
formed (malignant) cells. At the heart of this pro- 
tective system are the antigen receptor molecules 
found on B and T lymphocytes: the immunoglob- 
ulin (Ig) and the T cell receptor (TCR). Each 
lymphocyte expresses a distinct receptor mole- 
cule that confers on the cell a unique antigen 
specificity. The millions of different genes 
needed to encode these receptors are assembled 
from component gene segments by a site-specific 
process known as V(D)J recombination — so 
named for the V (variable) , D (diversity) , and J 
(joining) gene segments used in the reaction. 
V(D)J recombination is critical for the develop- 
ment of B and T lymphocytes and is the only site- 
specific recombination process know^n to occur 
in vertebrates. We are interested in two funda- 
mental questions concerning such recombina- 
tion: What is the biochemical mechanism of the 
reaction, and what molecular mechanisms regu- 
late the reaction during lymphoid development? 
This recombination reaction has been inten- 
sively studied since its discovery in 1976, yet lit- 
tle was known by the late 1980s about the enzy- 
matic machinery (recombinase) that carried it 
out. Particularly frustrating was the inability to 
identify the gene or genes encoding the V(D)J 
recombinase, despite a detailed understanding of 
the substrates and products. While working with 
David Baltimore, I developed a novel genetic ap- 
proach to the identification of these genes. Using 
a highly sensitive assay for V(D)J recombinase ac- 
tivity, I was able to demonstrate that gene transfer 
(transfection of genomic DNA) could activate the 
V(D)J recombinase in nonlymphoid cells, a sur- 
prising result implying that a single genetic locus 
was sufficient for the activation. 
Marjorie Oettinger and I then isolated this ge- 
netic locus and quickly encountered a second 
surprise. The locus contained not one but rwo 
genes, which functioned together to activate the 
V(D)J recombinase. Indeed, a mixture of these 
recombination-activating genes, called RAG-1 
and RAG- 2, was thousands of times more potent 
than was either gene alone. We then went on to 
demonstrate that the two genes are only coex- 
pressed in developing lymphocytes — in exactly 
those cells that are assembling Ig and TCR genes. 
This and a variety of other data suggest that RAG- 1 
and RAG- 2 encode the critical, lymphoid-specific 
components of the V(D)J recombinase. Interest- 
ingly, we found that RAG- 1 , but apparently not 
RAG- 2, is transcribed in neurons in the central 
nervous system, raising the possibility that RAG- 1 
has important roles in processes other than clas- 
sic V(D)J recombination. 
A central goal in our current research is to un- 
derstand the enzymatic mechanism of V(D)J re- 
combination, an elusive goal thus far, largely be- 
cause efforts to reconstitute the reaction in a 
cell-free system have been unsuccessful. As a first 
step toward this goal, we are studying the bio- 
chemical and enzymatic properties of the RAG- 1 
and RAG-2 proteins. Our initial focus is on devel- 
oping the necessary reagents — in particular, 
highly purified preparations of the proteins and 
antibodies that specifically recognize them. 
We will use these tools to examine the bio- 
chemical properties of the RAG proteins, asking 
whether they exhibit the activities expected of 
proteins involved in recombination (e.g., topo- 
isomerase, endonuclease, exonuclease, or ligase 
activities) . We are also interested in determining 
if the RAG proteins bind to DNA, either nonspecif- 
ically or by interacting with elements of the 
V(D)J "recombination signal sequence" [the 
DNA element required for V(D)J recombination]. 
In addition, we hope to ascertain whether the 
RAG-1 and RAG-2 proteins interact with one an- 
other or with other proteins and how these inter- 
actions vary during lymphoid development and 
among different cell types. 
Our second major focus is to understand the 
molecular mechanisms that regulate V(D)J re- 
combination during lymphoid development. The 
assembly of Ig and TCR genes is a complex and 
highly ordered process regulated in part at the 
level of expression of the V(D)J recombinase. Us- 
ing RAG-1 and RAG-2 as indicators, we are study- 
ing when and how the recombinase is turned on 
and then off again as B and T cells develop. Ex- 
periments performed in collaboration with Craig 
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