Genetic Mechanisms Involved in the Generation 
of the Antibody Repertoire 
Frederick W. Alt, Ph.D. — Investigator 
Dr. Alt is also Professor of Genetics and Pediatrics at the Children 's Hospital, Boston, and Harvard Medical 
School. He obtained his undergraduate degree in biology from Brandeis University and his Ph.D. degree 
in biological sciences from Stanford University, where he worked with Robert Schimke. He did postdoctoral 
work with David Baltimore at the Massachusetts Institute of Technology, after which he was Professor 
of Biochemistry and Microbiology at Columbia University College of Physicians and Surgeons. 
His honors include the Irma T. Hirschl Career Scientist Award, the Searle Scholars Award, 
and the Mallinckrodt Scholar Award. 
WE are interested in the molecular mecha- 
nisms that underlie the generation of a spe- 
cific immune response. The mammalian immune 
system functions through complex interactions 
between various cells and their products. Cells 
that effect specific immunological responses fall 
into two general categories: B lymphocytes that 
mediate humoral immunity (i.e., production of 
antibodies against foreign antigens) and T lym- 
phocytes that mediate cellular immunity (e.g., 
foreign graft rejection). During the earlier stages 
of lymphocyte development, stem cells proceed 
through a developmental program that ultimately 
leads to the generation of a multitude of individ- 
ual B or T lymphocyte clones (each clone is an 
essentially identical set of cells derived from a 
common parent). Each set of clonal cells ex- 
presses a novel receptor on its surface that will 
recognize a unique set of antigens. 
The ability of the immune system to respond 
specifically to a vast array of antigens results in 
substantial part from the unique organization of 
the genes that encode antigen receptor proteins. 
Unlike most genes, antigen receptor genes are not 
inherited intact from our parents. Instead, these 
genes are encoded in cassettes (gene segments) 
in the germline and are assembled into complete 
genes only during the somatic differentiation of 
lymphocytes. This lymphocyte-specific gene as- 
sembly process is referred to as VDJ recombina- 
tion. Because there are many individual cassettes 
that encode various portions of antibodies and 
because these can be put together in various com- 
binations or in various ways, the body can ran- 
domly assemble a vast array of different antibody 
genes from a limited amount of genetic material. 
Much of our work is aimed at determining the 
genetic mechanisms by which antibody genes are 
assembled from gene segments, the role of the 
gene assembly process in the generation of anti- 
body diversity, and the mechanisms that regulate 
this gene assembly process and ensure that it oc- 
curs only in appropriate cell types. We are also 
working on the elucidation of molecular signals 
that control the various steps of B lymphocyte 
differentiation. 
The enzymatic system involved in the assembly 
of antigen receptor genes involves a variety of 
different activities. Some of these activities are 
expressed specifically in developing lympho- 
cytes and are likely involved in the early pro- 
cesses of gene assembly, including recognition of 
the specific gene segments that will be joined and 
cutting them away from the surrounding genetic 
material . Other activities employed in VDJ recom- 
bination are likely expressed in many cell types 
where they may be involved in other processes, 
such as replication of genetic material or repair 
of lesions in genetic material (DNA repair) . The 
more widely expressed activities are probably re- 
cruited by the lymphocyte-specific components 
of the system to carry out certain aspects of the 
joining event, such as pasting together the "cut" 
gene segments. 
The lymphocyte-specific components of the 
VDJ recombination system are likely encoded by 
two genes (recently isolated by David Baltimore 
and David Schatz) referred to as recombination- 
activating genes 1 and 2 {RAG-1 and -2). Simulta- 
neous expression of these genes was found to oc- 
cur only in developing lymphocytes; artificially 
induced expression of the two genes (simulta- 
neously) in nonlymphoid cells confers them with 
the ability to undergo VDJ recombination. How- 
ever, these genes have been found to be ex- 
pressed in other tissues, including the central 
nervous system, fueling speculation that VDJ re- 
combination activity or the activity of one or the 
other of the two RAG gene products might be 
involved in developmental processes in tissues 
other than the immune system. 
To test unequivocally the function of the RAG- 
2 gene, we have used gene-targeting technology 
to eliminate a copy of this gene in mouse embry- 
onic stem cell lines. These cells were then intro- 
duced into developing mouse embryos, where 
they were incorporated into the germ cells of the 
resulting chimeric mouse. Mice that carried a sin- 
gle copy of the targeted mutation in their germ- 
line were interbred to generate animals that com- 
pletely lack the gene. Mice that lack the RAG-2 
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