MOLECULAR MECHANISMS IN LYMPHOCYTE DEVELOPMENT 
Stephen Y. Desiderio, M.D., Ph.D., Assistant Investigator 
The development of lymphocytes, the principal 
effector cells of the immune system, occurs in two 
phases. The first phase culminates in the deploy- 
ment of antigen receptors on the cell surface. The 
second phase, encompassing activation, growth, 
and terminal differentiation, is triggered in part by 
specific interactions between antigens and their re- 
ceptors. Molecular mechanisms that underlie both 
phases of immune development remain the focus 
of work in the laboratory. 
I. Molecular Mechanism of Antigen Receptor Gene 
Assembly. 
The variable regions of immunoglobulin (Ig) and 
T cell receptor (TCR) chains are encoded in sepa- 
rate germline DNA segments that are brought to- 
gether by site-specific recombination. Rearrange- 
ment is mediated by recombinational signal 
sequences— conserved heptamer and nonamer ele- 
ments that are separated by a spacer region. The 
spacer regions fall into two classes of 12 and 23 
base pairs (bp); recombination normally occurs 
only between gene segments carrying spacers of 
different lengths. Previous work in this and other 
laboratories identified B lymphoid cell lines that 
undergo continuing rearrangement of Ig gene seg- 
ments during propagation in culture. Dr. Desi- 
derio's laboratory subsequently developed a direct 
physical assay for the rearrangement of exogenous, 
integrated recombinational substrates in these cell 
lines; the assay has been used to examine the mo- 
lecular details of the recombination reaction and its 
DNA sequence requirements. In conjunction with 
this approach, the laboratory has undertaken to 
isolate components of the recombinational appara- 
tus by identifying and purifying proteins that inter- 
act specifically with recombinational signal se- 
quences. 
A. An alternative pathway of Ig gene rearrange- 
ment: mechanistic and immunologic implications. 
The assay for in vivo rearrangement of model sub- 
strates relies on direct observation of the products 
of recombination and places few constraints on the 
structures of the recombinants. This enabled Dr. 
Desiderio and his co-workers to discover a pathway 
for antibody gene rearrangement with important 
mechanistic and immunologic implications. This 
unusual reaction, which occurs at 1/10 the fre- 
quency of normal joining, results in the fusion of 
the recombinational signal sequences of one gene 
segment to the coding sequence of another, result- 
ing in signal sequence replacement. This observa- 
tion led Dr. Desiderio and his colleagues to pro- 
pose that in the recombination reaction the initial 
pairing and cleavage of Ig and TCR gene segments 
yields an intermediate, in which four free DNA ends 
(two coding ends and two flanking ends) are held 
in proximity Commonly this intermediate would be 
resolved by joining of the coding ends; less fre- 
quently the coding sequences of one segment are 
joined to the flanking sequences of the other, result- 
ing in signal sequence replacement. Signal sequence 
replacement can alter the targeting of Ig and TCR 
gene segments and represents an additional path- 
way for the generation of antibody diversity. 
B. Purification and characterization ofNBP, a pro- 
tein that specifically binds an enhancer of Ig gene 
rearrangement. Previous work in Dr. Desiderio's 
laboratory identified a protein in nuclei of lym- 
phoid cells and tissues that specifically binds DNA 
fragments containing Ig recombinational signal se- 
quences. In the past year the protein's DNA recog- 
nition site has been defined precisely, the in vivo 
function of this site has been examined, and the 
protein has been purified to homogeneity. 
To define the protein's binding site, a series of 
mutant substrates was generated. Mutations within 
the nonamer resulted in large (300- to > 1,000- 
fold) decreases in affinity; mutations outside the 
nonamer had at most a 10-fold effect. Thus the 
protein's recognition site coincides with the con- 
served nonamer. The protein has therefore been 
called NBP, for nonamer-binding protein. If specific 
recognition of the nonamer element plays a role in 
rearrangement, deletion of the nonamer would be 
expected to impair rearrangement. To test this, re- 
arrangement of wild- type or mutant recombina- 
tional substrates was assayed in vivo. Deletion of 
the nonamer element resulted in at least a 50-fold 
decrease in the frequency of Ig gene rearrange- 
ment, indicating that the nonamer is a profound 
enhancer of rearrangement. On the basis of its se- 
quence specificity, its preferential expression in 
lymphoid cells, and the impairment of rearrange- 
ment upon deletion of its binding site, it seems 
likely that NBP functions in Ig and TCR gene rear- 
rangement. 
Continued 
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