RESEARCH SUMMARY MARJORIE OETTINGER

The vertebrate immune system is capable of specifically recognizing and

responding to an enormous number of antigens. The interaction with antigen is

mediated by the immunoglobulin (Ig) and T cell receptor (TCR) molecules

expressed by B and T lymphocytes respectively. Much of the required diversity

in binding specificities is generated during lymphocyte development by a

process known as V(D)J recombination. Ig and TCR genes are encoded by multiple

segments of DNA that are not contiguous in the germ line but which are joined

together by the V(D)J recombination reaction.

We have isolated two recombination activating genes, RAG-1 and RAG-2, which

induce V(D)J recombination when expressed in mouse fibroblasts that normally

lack recombinase activity. These two genes are adjacent and are required

together to activate recombination in fibroblasts. While the RAG genes are

sufficient to induce recombination of an exogenously introduced reporter

construct, the fibroblasts endogenous immunogloublin and T cell receptor genes

are not subject to rearrangement.

The simultaneous expression of RAG-1 and RAG-2 correlates precisely with the

known expression pattern of V(D)J recombinase activity. Additionally, the two

genes are expressed independently from each other in certain tissues,

suggesting that they might have distinct functions individually: RAG-2, but

not RAG-1, is expressed in chicken B cells that are diversifying their

immunoglobulin repertoire by gene conversion. RAG-1, but not RAG-2, is

expressed in the mouse central nervous system.

RAG-1 and RAG-2 most likely encode the V(D)J recombinase, the

lymphoid-specific components of the V(D)J recombination machinery. My

laboratory is interested in understanding the activities of RAG-1 and RAG-2 in

V(D)J recombination and in other reactions in which they might participate.

Genetic and biochemical approaches are being employed to determine the

activities of the RAG proteins. We are carrying out mutational analyses of

RAG-1 and RAG-2. In addition, we are developing new assays for the V(D)J

recombination reaction in the hope of identifying individual steps in the

reaction and the factors responsible for those steps. Our ultimate goal is to

reconstitute the recombination reaction in vitro to facilitate its study in

greater detail.

While RAG-1 and RAG-2 are the only lymphoid specific genes whose expression is

required to induce recombination in fibroblasts, it is also clear that a

number of ubiquitously expressed factors are also involved. We have identified

three new genes that are expressed in all cell types based on their ability to

interact with RAG-1 in a yeast two hybrid assay. The affects of these genes,

Rch1, Rch2 and Rch3, on V(D)J recombination are now being explored.

In addition to studying the function of the RAG genes themselves and the

factors with which they interact, we are interested in studying the regulation

of recombination in lymphoid development. Thus, we are studying the regulation

of RAG expression during normal lymphoid development and in some mouse models

where there appear to be defects in RAG regulation. In addition, we are trying

to understand why introduced substrates are rearranged in RAG expressing

fibroblasts, while the endogenous genes can not be recombined.

A further interest of the laboratory is in trying to identify any human

diseases that might have a basis in defects in RAG-1, RAG-2 or any of the

factors with which they interact. The lymphoid cells of some human patients

with severe combined immunodeficiency syndromes have characteristics like

those seen in mice in which the RAG-1 or RAG-2 gene has been disrupted.

1. Schatz, D.G., Oettinger, M.A., and Baltimore, D. (1989). The V(D)J

recombination activating gene, RAG-1. Cell 59 1035-1048.

2. Oettinger, M.A., Schatz, D.G., Gorka C. and Baltimore D. (1990) RAG-1

and RAG-2, adjacent genes that synergistically activate V(D)J recombination.

Science, 248:1517-1523.

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