a ) T h e h e a v y ch a in locus (e x c lu d in g y, a an d s ex o n s)
Vn V2 V I ^ D n D 2 D l ^ J l J2 Jn ^ C |i [.is |im CÔ ÔS ôm
C K h - D O I K H H T
b)
V, D and J seg m en ts, sh o w in g th e ir re c o m b in a tio n re c o g n itio n seq u e n ces< Jl .2 3____ ^ 3 5' Vn V2 VI ^ Dn D2 D1 / / Jl J2 - c A ^ - c A — — 4% ^
%
t
D to J joint
Jnc) T he heavy chain locus after DJ jo in in g , and the excised intervening sequences 21,.I2 5' Vn V2 - c A — c A D2 J2 V to DJ jo in
d) T h e h e a v y chain locus a fte r V D J jo in in g , and th e ex c ise d in te rv e n in g seq u e n ces
5' Vn — o A V2 D2 J2 m w i N N .23,Jn + 3' VI Dn
one 12 base spacer and one 23 base spacer and precisely cuts the strands between the D and J segments and their heptamers. A circle of DNA is excised containing all the
intervening sequences. Coding sequence diversity is generated at the joint by loss of bases through imprecise joining, and by addition of bases by the enzyme Tdt. Tdt expression and its insertion of N regions occur only in murine bone marrow and not in fetal liver 47,107
The two heavy chain alleles appear to rearrange simultaneously and independently. Many murine pre-BI cells have undergone DJ rearrangement at both immunoglobulin heavy chain alleles. A study of human Epstein-Barr virus (EBV)-transformed lines shows that 19 in 25 have also undergone DJ joining at both alleles
There is evidence that D-D joining occurs in man, with both D segments becoming
incorporated into the heavy chain coding sequence This would provide a further
mechanism for expanding the immunoglobulin repertoire without increasing the size of the heavy chain locus. DIR segments have been described in the human genome that are similar to D segments, but have spacers of variable length It is possible that these segments could rearrange to conventional D segments without breaking the 23/12 base spacer rule. After DJ rearrangement, secondary rearrangements of upstream D segments to downstream J segments can occur This is a rather frequent event in vitro, but it is not known whether it occurs in vivo.
A V segment is then rearranged, to join to the rearranged DJ. Similarly, there is a heptamer/ 23 base spacer/ nonamer sequence 3' of each V segment and a nonam er/12 base spacer/ heptamer sequence 5' of D. Again, bases are lost and added at the join. The D segment and its flanking VD and DJ joints encode the CDR3 of the heavy chain antigen binding site. Sequence diversity in this region contributes to the generation of a broad repertoire o f antigen recognition. Over 90% of murine large pre-BII cells have at least one
VDJ rearrangement, and about 50% have rearrangement of both alleles After V to DJ
rearrangement is complete there are no remaining 12 base spacers in the locus and no further rearrangement can take place, except by an infrequent process of Vh replacement.
This uses a heptamer-like sequence within the rearranged V^ as an acceptor site for a 5' Vh segment
The heavy chain transcript includes V, D and J segments and the 4 Cp exons. If it terminates after the Cps mini-exon it results in a secreted form of p chain (ps). The membrane form pm includes the Cpm mini-exon that encodes a transmembrane domain and a short cytoplasmic portion. The Cp introns and the non-rearranged J segments are spliced out of the primary transcript. Human pre-B cell lines, unlike murine lines, produce 5m and ôs chains that are rapidly degraded ' ^ . Productive rearrangement that yields a full-length p polypeptide results in suppression of further rearrangement of the other heavy chain allele, a phenomenon termed allelic exclusion
1.3.2 Light chain gene rearrangement
Expression of ju polypeptide is generally followed by an increase in k light chain gene
rearrangement. The mechanism is similar to heavy chain rearrangement except that light chain genes do not contain D segments, loss of bases is less frequent, and no N sequence insertion takes place. The /c locus comprises alternative V and J segments, and a single C region. V and J segments of a light chain locus may have either a 12 base or 23 base spacer. Again, recombination is only possible between a segment with a 12 base spacer and one with a 23 base spacer.
Rearrangement of the X light chain gene on chromosome 22 generally follows k
rearrangement when the latter is unproductive. In man, the X locus comprises a cluster of VX segments followed by 7 CJX clusters, each containing one J and one C segment, as
shown in figure 1.3 Only 4 of the CJÀ clusters are fimctional Most human and
many murine B cells that express X have deleted their Jk and Ck segments This is
achieved by a rearrangement event using two recombination signals (RS), one of which is located in the Vk locus and the other downstream of Ck. RS k deletion is a common
event, but not a prerequisite for X gene rearrangement. The function of the deletion, if any, is unknown.
A ten-fold preferential usage of k over X light chain is observed in mouse. This bias is
already established at the level of rearrangement in small pre-BII cells