the vertebrate immune system employs a wide variety of antigen - specific receptors - the immunoglobulins and t - cell receptors - to recognize and neutralize foreign invaders . the receptor diversity necessary to recognize an almost limitless universe of potential pathogens is created by a site - specific dna rearrangement process termed v(d)j recombination . this unique reaction assembles the receptor genes from separate v , d and j gene segments , a process ostensibly restricted to lymphocytes at a certain stage of their development . in this view , the prediction is that the germline dna of a given organism should contain unrearranged receptor genes ; rearranged versions of the genes should exist only in lymphocytes . this prediction was satisfied by examination of germline and lymphocyte dna samples from a variety of familiar vertebrates , including mice , humans , birds , and common farm animals . the fly in the ointment , however , appeared with an analysis of receptor genes in the germline of evolutionarily distant organisms - the cartilaginous fishes . in these primitive vertebrates , many of the immunoglobulin genes are actually found pre - rearranged in the germline ( reviewed in ) . this suggests that the v(d)j recombinase may actually be an evolutionary force , a notion that is strongly supported by recent studies in the nurse shark . the evolutionary consequences of these site - specific germline gene rearrangements may reach far beyond the immune system . the recombination machinery recognizes dna sequences called recombination signal sequences ( rsss ) adjacent to each gene segment . each rss consists of conserved heptamer and nonamer motifs separated by 12- or 23-nucleotide ' spacer ' sequences . the recombinase is made up of two proteins , rag-1 and rag-2 , which , in conjunction with the non - specific dna - bending proteins , hmg-1 or hmg-2 , recognize the rss and catalyze site - specific dna cleavage ( see   for review ) . cleavage requires both rag-1 and rag-2 , and co - expression of these proteins is thought to be limited to developing lymphocytes ( , and reviewed in ) . as illustrated in figure 1 , the rag proteins introduce a double - strand dna break precisely between the v , d or j coding sequence and the rss , generating two types of dna ends : blunt signal ends ( which terminate in the rss ) and covalently sealed ( hairpin ) coding ends ( which terminate in the v , d or j element ) . after cleavage , the two signal ends are joined , producing a signal joint . prior to joining the coding ends , the hairpins must be opened ; joining generates a coding joint that may have lost or gained nucleotides . the precise details of the end - processing and joining reactions remain obscure , and are not important for this story ; it is clear , however , that multiple , non - lymphoid - specific dna repair proteins are involved . one detail that is important is that the opening of the hairpins frequently occurs ' off center ' , leading to palindromic single - stranded tails . joining of these ends can give rise to a characteristic signature in the completed junction : a palindromic , or p nucleotide , insertion . another type of junctional insertion , n ( non - templated ) nucleotides , are added randomly by the enzyme terminal deoxynucleotidyl transferase . thus , coding joints formed by v(d)j recombination have several distinguishing characteristics , including variable loss of nucleotides and the frequent presence of either n nucleotides , p nucleotides , or both . v(d)j recombination bears many striking parallels to the movement of certain transposable elements , both in its general form and in important mechanistic details of the reaction ( reviewed in ) . in fact , recent biochemical experiments have shown that purified rag proteins can catalyze transposition in the test tube , integrating a dna fragment bearing signal ends into a target duplex ( figure 2 ) . this reaction does not require specific dna sequences in the target and , like many transposition reactions , creates a characteristic ' footprint ' : upon integration , three to five nucleotides of target dna are duplicated on either side of the transposon . it should be noted that there is , as yet , no firm evidence that rag - mediated transposition events can occur in living cells . early on , it was suggested that the v(d)j recombination system might have arisen by the fortuitous integration of a transposable element into an ancestral antigen - receptor gene . this hypothesis was strengthened by the discovery that the rag genes are tightly linked , and by the finding that the rag proteins can act as a transposase . thus , a plausible model for the acquisition of the v(d)j recombination system during vertebrate evolution is the integration of a transposable element carrying the linked rag genes into a primordial antigen - receptor gene in an ancestral jawed vertebrate , approximately 450 million years ago ( reviewed in ) . presumably , this initial integration event created the first rearranging antigen - receptor gene ; subsequent gene duplication events then created the multiple immunoglobulin and t - cell receptor loci . to learn more about the evolutionary origins of the combinatorial immune system , several laboratories have characterized antigen - receptor loci from a wide variety of species , including the cartilaginous fishes - the living jawed vertebrates most phylogenetically distant from mammals . the immunoglobulin loci of sharks and skates contain some fairly typical antibody genes , with multiple v , d and j elements . surprisingly , however , many of the immunoglobulin genes are already partially or fully rearranged in the germline ( see   for review ) . recently , pre - rearranged immunoglobulin genes were also found in a teleost fish , the channel catfish . one possibility is that they are descendants of the ancestral antigen - receptor gene , before integration of the putative transposable element . a second possibility is that these genes arose from rag - mediated dna rearrangement events that occurred in the germline , an operation that violates the precept that the rag recombinase is functional only in developing lymphocytes . a recent paper by lee et al .   addresses these questions by examining immunoglobulin genes in the nurse shark . the nurse shark ns4 immunoglobulin light chain gene family provided very useful information , as there are several highly homologous genes present both in pre - rearranged and unrearranged forms . these features allowed the authors to evaluate sequences in sufficient detail to ascertain whether the genes bear characteristic features of v(d)j recombination or footprints of transposition . their analysis revealed that the pre - rearranged genes did indeed contain tell - tale signs of coding joints formed by v(d)j recombination , including both n nucleotides and p nucleotides , which strongly suggest a hairpin intermediate . the presence of these features in several junctions suggests several independent germline v(d)j recombination events , although analysis of multiple unrelated individuals suggests that these events are not frequent . importantly , analysis of the unrearranged ns4 genes failed to detect the target site duplications that are hallmarks of transposon insertions . thus , while the pre - rearranged ns4 genes appear to have been derived from unrearranged genes by germline v(d)j recombination , there is as yet no evidence to support the hypothesis that the unrearranged genes were derived from the pre - rearranged genes by insertion of a transposable element ; this is discussed in a recent review by lewis and wu . the recent studies of the nurse shark ns4 genes strongly suggest that v(d)j recombination events can occur in the germline . moreover , phylogenetic analysis indicates that these events occurred recently ( at least from an evolutionary perspective ) , some time within the last 7 million years . what evolutionary benefit might there be in germline recombination events ? pre - rearranged immunoglobulin genes may confer certain advantages over genes that must be assembled by recombination in individual lymphocytes . for example , pre - rearranged genes may encode receptors capable of recognizing common pathogens likely to be encountered during the neonatal period , before the development of a full repertoire of rearranged antigen receptors . furthermore , germline joining could have contributed to evolution of gene segment clusters , and possibly the evolution of d segments . to my knowledge , co - expression of both rag proteins outside the lymphoid system has not been reported , but rna species encoding rag-1 and rag-2 have been detected in zebrafish ovary and xenopus oocytes , respectively . even if the recombinase is not normally expressed in these tissues , though , inappropriate expression might occur occasionally , perhaps as a result of improper reprogramming of tissue - specific gene expression during development . if rag expression does occur during the development of germ cells , another important question arises : how are loci chosen for rearrangement ? during normal b - lymphocyte differentiation , immunoglobulin loci undergo a carefully orchestrated series of rearrangements . d to j rearrangements of the heavy - chain genes occur first , followed by v to dj rearrangements , followed in turn by rearrangement of the light - chain genes . the carefully ordered sequence of rearrangements is critical for proper lymphocyte differentiation and is thought to be controlled by accessibility of the loci , mediated by alterations in chromatin structure ( reviewed in ) . rsss present in antigen - receptor loci that have not been ' targeted ' for rearrangement are used rarely , if at all . the careful control of locus accessibility seen in lymphocyte differentiation may not be recapitulated in the development of germ cells - after all , these cells are not supposed to be expressing rag recombinase activity . furthermore , the v(d)j recombinase is not particularly picky about the sequences it can target for rearrangement . lewis and co - workers have found that ' cryptic ' sites capable of supporting rearrangement occur at least once in every 600 base pairs of a commonly used plasmid sequence , and they have estimated that there are at least 10 million such sites in the mammalian genome . in fact , there is evidence that cryptic sites in the mammalian genome can serve as targets for rag - mediated rearrangement in lymphocytes ( reviewed in ) . thus , it is possible that rag expression during germ - cell development might cause rearrangements of regions of the genome far removed from the immune receptor loci . these considerations suggest that the v(d)j recombinase might have been ( and could still remain ) a significant force shaping vertebrate evolution , by catalyzing v(d)j - like rearrangements and , perhaps , transposition . comparison of genome sequences from a variety of organisms may allow some aspects of this notion to be tested . first , the rag proteins bind to the rsss ( triangles ) and bring them together into a synaptic complex . cleavage ensues , generating a pair of blunt signal ends and a pair of dna hairpin coding ends . joining of these ends generates signal and coding joints , respectively . a fragment of dna generated by rag - mediated cleavage , with rag proteins bound to the signal ends ( the donor ) , can capture another dna duplex ( the target ) . the rag proteins bound to the signal ends catalyze integration into the target , generating a characteristic duplication of the target sequence at the integration site ( arrowheads ) . i thank vicky brandt for editorial assistance and mark landree for critical comments on the manuscript . work in the author 's laboratory is supported by the howard hughes medical institute and by grants from the national institutes of health , the human frontiers science program , and the american cancer society .
the antigen - receptor genes of vertebrates are rearranged by a specialized somatic recombination mechanism in developing lymphocytes - and , unexpectedly , also in the germline of cartilaginous fishes . 
 the recombination system that carries out these dna rearrangements may thus be a significant evolutionary force , perhaps not limited to rearrangements at antigen - receptor loci .