Molecular Mechanisms of Lymphocyte Differentiation 
antibody gene assembly. Rearrangement of anti- 
body and T cell receptor genes is mediated by 
conserved DNA sequences (signal sequences) 
that lie next to the gene segments, near the sites 
of recombination. It appears that during the ini- 
tial stages of rearrangement, two antibody seg- 
ments are paired and then cleaved to form an in- 
termediate in which four DNA ends (the ends of 
the gene segments and the ends of their flanking 
signal sequences) are held near each other by the 
recombinational machinery. One of our aims is to 
test this idea by recovering such intermediates 
from B cells that carry model recombinational 
substrates. 
Essential to an understanding of B and T cell 
development is a molecular description of anti- 
body and T cell receptor gene rearrangement. 
Our approach to this problem began with the 
guess that the recombinational signal sequences 
represent parts of a scafi'old on which the recom- 
binational machinery is assembled. We examined 
extracts of immature B and T cells for proteins 
that could bind specifically to these signals. Our 
search uncovered a specific DNA-binding protein 
whose target coincides precisely with one of the 
recombinational signals: a conserved 9-base pair 
(bp) DNA segment that is needed for efficient 
rearrangement of antibody genes. At the same 
time, other laboratories identified at least three 
genes whose products likely play a role in rear- 
rangement. We are now examining the relation- 
ship between these gene products and the DNA- 
binding protein that we have purified, with the 
goal of understanding what the recombinational 
machinery is and how it works. 
Lymphocyte Activation 
A second area of work in the laboratory ad- 
dresses a different problem: the processes by 
which specific antigen and growth factors trigger 
the activation of B and T cells. We have set out to 
identify molecules that participate in these pro- 
cesses, with the goal of understanding how these 
molecules function in the generation of an im- 
mune response. We have begun by focusing on a 
group of enzymes called tyrosine kinases. Bio- 
chemical evidence has long suggested that these 
enzymes might be intimately involved in B and T 
cell activation, but the molecules themselves 
have proven elusive. Among genes expressed in 
interacting B and T cells we have found one that 
encodes a novel tyrosine kinase that is homolo- 
gous to the cellular proto-oncogene c-src. Unlike 
c-src, however, this gene shows a strikingly re- 
stricted pattern of expression, being transcribed 
preferentially in B cells and in developmental 
precursors of B cells. We have accordingly called 
this gene blk (B lymphoid kinase) . The blk gene 
is activated early in B cell development and is 
expressed along with several proteins that are 
known to transmit signals across the B cell mem- 
brane, including the antibody heavy chain. When 
B cells develop into antibody-secreting cells or 
plasma cells, they no longer express these signal 
transmission proteins and also stop expressing 
blk. This suggests that the product of the blk gene 
interacts with a receptor that spans the B cell 
membrane and that senses the presence of anti- 
gen or a specific growth signal. 
How might extracellular signals activate the 
blk kinase? Our recent results suggest that the blk 
kinase is inactivated by addition of a chemical 
group to a specific site on the kinase molecule; 
the enzyme is activated when this chemical 
group is removed. If the target site on the blk 
kinase is mutated so that it cannot accept the in- 
activating chemical group, the enzyme is locked 
into the "on" state. Expression of this mutant ki- 
nase in certain types of cells leads to alterations in 
growth. By concentrating on the specific extra- 
cellular signals that turn the blk kinase on, we 
hope to identify the pathways in which blk 
functions. 
Aside from its likely role in B cell signaling, blk 
provides a window into how gene expression is 
restricted to specific cell types during develop- 
ment. To date, most work on regulated expres- 
sion in B cells has focused on the immunoglobu- 
lin genes. The pattern of expression of blk among 
B cell lines differs from the pattern of expression 
of immunoglobulin genes. The blk gene occu- 
pies approximately 30 kbp of DNA on mouse 
chromosome 14. Its unusual structure carries 
two transcriptional promoters. One of these is 
preferentially used early in B cell development, 
while both promoters are used at later stages. We 
expect that this temporal regulation reflects dif- 
ferences in the arrays of DNA-protein complexes 
that drive transcription of blk in immature and 
mature B cells, and we now wish to explore these 
differences in detail. 
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