MECHANISM AND CONTROL OF THE ASSEMBLY OF ANTIGEN-RECEPTOR GENES 
Frederick W. Alt, Ph.D., Investigator 
Dr. Alt's laboratory is defining molecular aspects 
of the development of antibody-producing cells, 
with a focus on the mechanism and control of the 
genomic recombination events involved in this dif- 
ferentiation process. 
Activities Involved in VDJ Recombination 
Genes that encode the variable regions of immu- 
noglobulin (Ig) and T cell receptors (TCRs) are as- 
sembled from germline variable (V) , diversity (D) , 
and joining (J) gene segments during precursor lym- 
phocyte differentiation. All Ig and TCR gene seg- 
ments are assembled by a common, lymphoid- 
specific activity referred to as VDJ recombinase. 
The VDJ recombination mechanism involves the 
recognition of conserved recombination sequences 
(RSs) that flank each germline V, D, or J segment, 
the introduction of double-stranded breaks at the 
RS/coding sequence junctions, the potential loss 
and/or addition of nucleotides at the coding junc- 
tions, and polymerization and ligation activities to 
complete the process — usually resulting in joining 
of the two participating RSs (RS joins) and in joining 
of the coding segments (coding joins) . By analogy to 
other systems, the VDJ reaction could be orches- 
trated by one or two activities that confer specific- 
ity, while other events (e.g., ligation) might be ef- 
fected by ubiquitous cellular activities recruited to 
function in VDJ recombination. 
Tissue-specific activities. The recombination- 
activating genes 1 and 2 (JRAG-l and -2) were iso- 
lated by others, based on an ability to confer VDJ 
recombination activity to nonlymphoid cells syner- 
gistically. To analyze RAG function in normal lym- 
phocytes. Dr. Alt's group used gene-targeted muta- 
tion in embryonic stem (ES) cells to generate mice 
that carry a nonfunctional RAG- 2 gene in their 
germline. Mice homozygous for the RAG-2 muta- 
tion do not generate mature B or T cells but other- 
wise appear normal — indicating that RAG-2 func- 
tion (and VDJ recombination) is required only for 
lymphocyte development. Work in the laboratory of 
Dr. Susumu Tonegawa (HHMI, Massachusetts Insti- 
tute of Technology) has led to similar conclusions 
regarding RAG 1 function. 
Rag-2 mutant animals accumulate B and T cell 
progenitors that have not rearranged their Ig or TCR 
loci. Pre-B cell lines derived from these mice do not 
have rearrangements of endogenous antigen recep- 
tor loci; however, they rapidly rearrange their Ig 
heavy-chain locus upon introduction of RAG-2 ex- 
pression vectors. Thus B and T cell development in 
iMG-2-deficient mice is blocked due to an inability 
to initiate VDJ recombination. 
Ubiquitously expressed activities. Mice homozy- 
gous for the scid (severe combined immune defi- 
cient) mutation {Scid mice) also have impaired abil- 
ity to generate mature B and T cells. Dr. Alt's 
laboratory and others demonstrated that pre-B cells 
in these mice initiate VDJ recombination correctly 
(including introduction of precise double-stranded 
breaks) and also form RS joins relatively normally. 
They are impaired, however, in their ability to form 
coding joins. Unlike the RAG-2 mutation, the scid 
mutation is "leaky," in that Scid mice develop ma- 
ture lymphocytes as they age. This leakiness results, 
at least in part, because liberated coding ends occa- 
sionally can be joined by illegitimate recombination 
to form coding joins. Because RAG-deficient animals 
cannot initiate VDJ recombination, their defect is 
not leaky. 
Others found that both lymphoid and nonlym- 
phoid cells of Scid mice appear to have a defect in 
DNA repair. Thus the overall phenotype of the scid 
mutation suggests that it may affect a more generally 
expressed factor recruited to perform one of the ter- 
minal events of VDJ recombination. 
To elucidate further the potential relationship be- 
tween VDJ recombination and DNA repair. Dr. Alt's 
group tested a large number of existing mutant Chi- 
nese hamster ovary (CHO) cell lines with defects in 
either excision repair or double-strand break repair 
(dsbr) for an ability to rearrange introduced VDJ 
recombination substrates following introduction of 
constitutive RAG expression vectors. All excision 
repair mutants showed normal VDJ recombination; 
however, 3 of 5 tested dsbr mutants were markedly 
impaired in this process. Two (xrs-5 and XR- 1 ) were 
impaired in an ability to form both coding and RS 
joins while another (V-3) was impaired primarily in 
coding but not RS joining (similar, if not identical, 
to the impairment generated by the murine scid 
defect) . 
Because each CHO mutant belongs to a different 
complementation group, their defects are likely en- 
coded by different genes. In this regard, xrs-6 and 
XR-1 lines that had reverted to normal their DNA 
repair defect were isolated by others via introduc- 
tion of specific human chromosomes. These lines 
also reverted to a normal ability to undergo VDJ re- 
combination. Together the findings indicate that 
IMMUNOLOGY 303 
