Cell, Vol. 63, 967-997,

November

30. 1990, Copyright

0 1990 by Cell Press

Somatic Diversification of lmmunoglobulin Heavy Chain VW Genes: Evidence for Somatic Gene Conversion in Rabbits Robert S. Becker’ and Katherine L. Knight Department of Microbiology and Immunology Stritch School of Medicine Loyola University Chicago Maywood, Illinois 60153

Rabbits preferentially utilize only one of their multiple functional germline immunoglobulin VH genes. This preferential usage of one gene, VH7, miser, the question of how rabbits generate antibody dlvemlty. VDJ divemification was analyzed by cloning and sequencing VH1 gene rearmngements. Comparison of these sequences with that of germline VH1 identlfled clusters of nucleotlde changes, including codon insertlons and deletions. To Investigate whether gene conversion was Involved in this somatic divemiflcatlon, we -arched a data base of mbblt germline VH gene sequences for donor VH genes; potentlal donors were identified for five dlvemlfied reglons. We conclude that somatic gene commion has a maJor role In generating antlbody di~mlty in rabbits. These studles provide clear evidence for somatic gene convemion of mammalian VW genes. Introduction Vertebrates produce a diverse array of antibodies whose V regions specifically bind to one of a large number of antigens. In mammals, antibody diversity is generated through combinatorial joining of multiple V, D, and J heavy chain gene segments and multiple V and J light chain gene segments. Additional somatic diversification in V regions occurs by hypermutation (reviewed in Kocks and Rajewsky, 1969) at a rate estimated to be lop3 per base pair per cell generation (McKean et al., 1964). Chickens, like mammals, have multiple heavy and 1 light chain V gene segments, but unlike mammals, chickens use only one of the VH and one of the Vi gene segments in heavy chain VDJ and light chain VJ gene rearrangements, respectively (Reynaud et al., 1965, 1969). The remaining VH and VI genes in chickens are pseudogenes and are used to diversify the functionally rearranged VDJ or VJ gene by gene conversion (Reynaud et al., 1987; Thompson and Neiman, 1987). Although Maizels (1969) suggests that gene conversion plays a significant role in the generation of antibody diversity in mice, the prevailing sentiment is that most of the somatic diversification in mammals is due to somatic mutation rather than to gene conversion (Chien et al., 1966; Kocks and Rajewsky, 1969; Wysocki and Gefter, 1969). The rabbit heavy chain chromosomal region is organized similarly to that of other mammals in that clusters * Present

address:

Connaught

Laboratories,

Swiftwater,

&nn+ania.

of V, D, and J gene segments are found on a single chromosome with the V and J gene segments being separated by 63 kb of DNA (Becker et al., 1969). We have identified three families of germline D gene segments, Dl, D2, and D3, and five JH gene segments (Becker et al., 1989). Although four of the JH gene segments are functional, one, J&, is utilized in most VDJ rearrangements. We believe there are a few hundred germline VH genes (Gallarda et al., .1986); approximately 100 have been cloned (Currier et al., 1988). Clusters of the 3cmost VH genes were cloned from rabbits homozygous for each of the VHa heavy chain alleles, al, a2, or a3 (Becker et al., 1989; Knight and Becker, 1990). The 3’-most VH gene of each V,+a chromosome is designated l/,7; the six genes immediately upstream of V,7 are designated VH2 through V,.,Z For the a3 chromosome, nucleotide sequence analyses of the seven 3’-most VH genes indicate that four (V~7-a3, V+a3, V,.& a3, and V,.&a3) appear functional, while the other three (VH2-a3, V&-a3, and V”793) are pseudogenes. Among mammals studied thus far, rabbits are unusual because they preferentially utilize one VH gene, V,7, in their VDJ rearrangements (Knight and Becker, 1990). We based the conclusion of preferential VH use on several observations and deductions. Greater than 80% to 90% of rabbit serum lg molecules bear VHa allotypic specificities, al, a2, or a3 (Dray et al., 1963). The &+a allotypes behave as if controlled by alleles. One might expect that these allotypes could be inherited in an allelic manner if only one or a few closely linked VH genes are utilized in VDJ rearrangements. We have shown that the 3’-most VH gene, VH7, from the al, a2, or a3 chromosome encodes the al, a2, and a3 allotype, respectively (Knight and Becker, 1990), and we have also shown that the VH gene used in each of eight VDJ rearrangements in leukemic B cells was V,7, irrespective of VHa allotype (Becker et al., 1990; Knight and Becker, 1990). From these studies, we suggested that V,7 was preferentially utilized in VDJ rearrangements and that the preferential utilization of these alleliC VHa allotype-encoding V”7 genes would explain both the presence of VHa allotypeS on mOSt (80% to 90%) serum lg molecules and the allelic inheritance pattern of the VHa allotypes. This idea was confirmed by results of molecular genetic analysis of the mutant rabbit Alicia (Kelus and Weiss, 1986); on the a2-derived A/i chromosome a 10 kb deletion resulted in the loss of V,7 and a concomitant loss of &.,a2 allotype expression (Knight and Becker, 1990). We obtained additional evidence that V,7 is preferentially utilized in VDJ rearrangements from Southern analysis of polyclonal B lymphocyte DNA. By analyzing Hindlllrestricted peripheral blood leukocyte (PBL) DNA from adult rabbits, we identified a rearranged JH -hybridizing fragment of 4.1 kb, the size expected if V,7 had been rearranged to the J& gene segment (Knight and Becker, 1990). Preferential utilization of one VH gene in VDJ rearrangements indicates that random combinatorial joining of multiple VH to DH and JH gene segments is not a major

WI 988

VHZ-a3 phl.1

-a3

BK

VDJ HHEH

Figure 1. Restriction Map of VDJ Phage Clone l.l-a3 and of Germline V,.,7-a3 and V&a3 Genes from Cosmid Clone Cos 9.2

CP

BSaSEBHE

H

HB

X

Solid boxes, VH gene segments; open boxes, JH gene segments; hatched box, Cs. B, BamHI; K, Kpnl; H. Hind III; E, EcoRI; X. Xhol: Sa, Sall; S, Sacll (Becker et al., 1989)

I

V&a3 Germline

a3

VHl -a3

BK

HHEH

contributor to the generation of antibody diversity. We now face the question as to how antibody diversity is generated in rabbits. To understand how antibody diversity is generated, we must compare the nucleotide sequences of diversified V

genes with their germline homologs. Since germline V,7 is preferentially utilized in rabbit B lymphocytes, we can expect that most VDJ rearrangements cloned from these cells will utilize V,7. In this study, we cloned and analyzed VDJ rearrangements from DNA of spleen and lymph

Figure 2. Comparison of Nucleotide quences of VH7-Utilizing VDJ Genes and MLN-a3 with Germline V&a3

Noncoding sequences are shown in lowercase. Amino acid numbers follow that of Kabat et al. (1987). Dn and JH regions are marked. Dashes designate nucleotides identical to Vnla3; slashes represent the absence of nucleotides. The beginning of the leader intron is marked Li. o = region from which sequence for PCR primer was derived.

VHl-a3 l.l-a3 HLN-a3

aactttttaaaataatggtaacataaaattcattcatatgatctgaatcctatcacagccatcactctacatacccaggacac _________._._._._.__----.-.~.-.-------.-..-.-..--.-.--.-----.-..-.-------.--.... _____________..._._.-.--.-.-..---.----.-..-...-...----.-----.-.--.-.-----.-....-

Vtll-a3 1.1-83

CaCatCtgCCCtgggCCCtCtcctgtctgaggcgtctacctcatgcctgctatataggggcagcac~tgcaaat~gggcc _._________._______.________.....__.____._...._____._.________._..~.~~~~~~~.....

VHl-a3 1.1.a3 "LN.AJ

tccctctgccc~tgaaaaccagcccagccctcaccctgcagctctggcacaggagctccagccccagcactcccaggtgt ._.._.______..._____-----.-.-..-.-.--.-..-.-..-..-.--.--.-.-----.--.----.--.-... . . ..-.-___________.....-~.~.~...._._...._.._.._.___~~.~....~~.~~.~..~.~...~.~..~

VHl-a3 1.1.a3 fjLNwa3

ccactcagtgatcgcactcaacacagacactcacc .-.-\--.-.-.-..------------.-....-. . ..-.-.__._._____._-----~~~~~.~.~..

VHl-a3 1.1.a3 "LN.a3

-5 Li GCT GTG CTC AAA G gtaatgatggagaaCgCggggcactgagtCtggg~~~~~aCgtgagt~~~~~~C~CagaCag ___ __. ___ _.. _ ---.-------‘-.-.-.--.-.-----..-.~----.--.-.---.-.~~.--~-.--g-. .__ __. . . . .__ _ _._.___.._.____._._..-.--....--.-------.-..-~..-.--.-.--.--...

VHl-a3 1.1.a3 "LN.a3

tgtgagtgacag\tacctgaccatgtcgtctgtgttttcag .___________ C-t---.-.--.-.-f----.----g... .-.--.------\-.-.-.-.--------------.-g-..

VHl-a3 l.l-a3

10 20 TCC GGG GGA WC CTG GTC AAG CCT GGG GCA TCC CTG ACA CTC ACC TGC ACA CCC TCT CGA .__ --- ___ -G- __. .__ c.- ___ -A. -G- . . . .__ __. _._ ___ _.. .__ __. .-_ _..

HLN-a3

.--

VHl-a3 l.lea3 nLN.a3

IDRl 32 35A FRZ 40 TTC TCC TTC AGT AGC AGC TAC TAC ATG TGC TGG GTC CGC CAG GCT CCA CCC AAG GGG CTG .-. .-- ___ ___ ___ .-- ._. -GG -.A ._. _._ ._. ___ .__ _.. _._ ._. __. . . . .._ -.- .-. ___ __. _._ G-- ._. _.. _._ _.. ._. _._ ._. . . . .._ ._. . . . .._

VHl-a3 1.1.a3 MLN-a3

CDRZ 52A 528 60 GAG TGG ATC GCA TGC ATT TAT GCT GGT ACT AG, GGT AGC ACT TAC TAC CCC AGC TGG GCG .__ _.. ._. ___ ___ ___ ___ .G- _._ ___ G.- . . . TA- __. ___ _._ _._ .c- _._ _._ .-. ._. ___ ___ ___ G-. .T- _._ ___ ___ -T. ___ __. ___ ___ _.. .__ ___ ._.

VH1-a3 1.1.a3 MLN-a3

FR3 AAA GGC CGA TTC ACC ATC --- -.- __. --A ___ --- -.- __. ._. .__ ..-

VHl-a3 1.1.a3 HLNea3

a28 AGT -----

1.1.a3 MLN-a3

Jll ACT ACT TAT TAT CAT \\\ TTT AAC TTG TGG CCC CCA CAA \\\ \\\ \\\ \\\ CCC TTT AAC TTG TGG GGC CCA

-19 ATG GAG ACT GGG CTC CCC KG CT1 CTC CTG CTC .__ .__ ___ _._ .__ ___ ___ .._ ___ ___ .-- ._. ._. _.. . . . ._. ___ .__ _.. .-_ _._

LilOO

---

___

.__

._.

.--

_._

___

__.

..-

_..

-3 CT GTC CAG TGT ._ .__ _.. .__ ._ _._ . . . .--

.__

._.

. . .

Li55

1 3 CAG \\\ KG TTG GAG GAG __. GAG CA- c.- .T. ___ __. \\\ G+- -.. ._. ___

-__

_._

.A-

. . .

. . .

___

70 80 a2A TCC MA \\\ ACC TCG TCG ACC ACG GTG ACT CTG CM ATG ACC ___ ._. \\\ _._ ___ _.. .__ __. .__ ___ _._ . . . . . . ._. A-- -G- AGC _._ p&c CT- .A. ___ . . . _._ _._ __. c.. .A.

a2c CTG ACA CCC GCG GAC ACG GCC ACC -.- __. ___ .__ ._- ___ __. ---.- __. __. .-. --- ___ ___ ._.

SeI.&a3

90 DN TAT TTC TGT GCG AGA GA --- --. --- --C --- \\\ CCA TAT ACG AGT GGT .._ _.. _._ ._. .._ .GT TAT GCT GGT *(-g AGT

Somatic 999

Gene

Conversion

of lmmunoglobulin

VDJ Genes

node cells from homozygous a9’a3 adult rabbits. By comparing the nucleotide sequences of the diversified VU+ utilizing VDJ genes to germline V,7, we identified clusters of nucleotide changes in framework regions (FR) and in complementarity determining regions (CDR). Based on our observations, we propose that rabbits, like chickens, use gene conversion as one mechanism for generating antibody diversity. Results Somatic Diversification of VH1 in Rabbit VW Rearrangements To analyze somatic diversification of rabbit VDJ rearrangements that had utilized VH7, we cloned VDJ genes from genomic DNA of mesenteric lymph node (MLN) and spleen from adult a3/a3 rabbits. Southern analysis of one VDJ clone, phl.1, obtained from the recombinant phage library constructed from DNA of rabbit #207A3, showed that it contained one germline VH gene, one VDJ gene, and the C~I gene (Figure 1). The restriction map of phl.1 was compared with that of a cosmid clone containing germline VH7-a3 and VH2-a3; the restriction sites 5’ of the VDJ gene of clone phi.1 and 5’ of germline VH7-a3 were found to be identical. The germline VH gene of phl.1 was located at the same position as germline VH2-a3. These data indicated that V,7 was used in the VDJ rearrangement contained in clone phl.l and that the upstream VH gene in phl.1 was probably VH2-a3. To prove this, we determined the sequence of nucleotides of the BamHI-Kpnl fragment (308 bp; Figure 1) of this gene and showed that it was identical to that of the previously sequenced germline VH2-a3 gene (Knight and Becker, 1990; sequence submitted to GenBank). From this evidence, we concluded that the upstream gene was VH2 and that the gene utilized in the VDJ rearrangement of phl.1 was indeed VH7. The nucleotide sequence of the VDJ gene of clone phl.1, hereafter designated l.l-a3, was determined; the sequenced fragment began 515 bp 5’ of the ATG start codon and extended to the BstEll site (Becker et al., 1989) in the JH segment (Figure 2). With the exception of the regions bounded by nucleotides 55 and 100 of the leader intron and by codons 2 and 16 of VH, the sequence of l.la3 was nearly identical to germline VH7-a3 (870 of 882 bp). The region of difference bounded by VH codons 2 and 16 was unusual in that it contained eight nucleotide differences plus an insertion of a codon at position 2. One nucleotide change in Vn codon 16 resulted in the appearance of a BamHl restriction site, not present in germline V,.,7-a3. Previous data indicated that the extra codon at VH position 2 is found in approximately 50% of germline VH genes (Currier et al., 1988), and this suggested to us that the sequence for the l.l-a3 variant region bounded by VH codons 2 and 16 may have been derived from a different germline VH gene. To find such a gene, we searched our data base of nucleotide sequences of germline VH genes from the a3 chromosome (Knight and Becker, 1990; Currier et al., 1988; Bernstein et al., 1985). Of the 11 available germline

genes examined, one, VH6-a3, had a nucleotide sequence for VH codons 2 through 16 identical to that of l.la3 (Figure 3A). Thus, we conclude that a gene such as V,&a3 could have donated a nucleotide sequence including Vu codons 2 through 16 to the rearranged VH7-a3 gene in clone phl.1. Similarly, we searched the 11 germline VH genes for the sequence of nucleotides 55 through 100 of the leader intron in l.l-a3, and except for position 55, identical sequences in germline VH3-a3 and V&-a3 were found (Figure 38). We conclude that a gene such as V,.,3-a3 or V&-a3 could have donated leader intron sequences to l.l-a3. Thus it appears that at least two regions of the V,7 gene in l.l-a3 have been donated by other germline Vu genes. The three nucleotide differences in CDRl, codons 34 and 35, of l.l-a3 are found in germline V,.&-a3, and we suggest that this may represent a third region of l.l-a3 modified by intergenic transfer. A second VDJ rearrangement was cloned from a Charon 28 phage library of DNA from rabbit #18485. The restriction map of this clone, MLN-a3, was compared with the restriction map of the germline VH7-a3 and with the map of the VH7-a3-utilizing VDJ rearrangement in clone 41-3, described previously (Figure 4; Knight and Becker, 1990; Becker et al., 1990). This comparison revealed that the region upstream of the MLN-a3 VH gene was identical to the region upstream of the V,.,I-a3 and 41-3 VH genes, indicating that MLN-a3 utilized VH7-a3 in its VDJ rearrangement. To confirm this, we isolated the 1.5 kb Hindlll-Ncol fragments 5’ of Vu from MLN-a3, VH7-a3, and 41-3 (Figure 4) and restricted them with Ddel, Haelll, and Hinfl; polyacrylamide gel electrophoresis showed the restriction fragments of MLN-a3 to be identical in number and size to those of l/,.,7-a3, 41-3 (Figure 5) and the V,+7utilizing VDJ l.l-a3 (data not shown); these data indicate that the VDJ rearrangement in clone MLN-a3 utilized V,.,7-a3. As a negative control, these fragments were compared with those generated from the 1.5 kb Hindlll-Ncol fragment of the V&a3 germline gene, a gene similar to, but distinct from, VH7-a3 (Figure 4). The restriction fragments of MLN-a3 showed no similarity to those of V&-a3 (Figure 5). From these data, we conclude that MLN-a3 utilized VH7-a3 in its VDJ rearrangement. We sequenced the MLN-a3 VDJ rearrangement from a site 515 bp 5’ of the ATG start codon to the BstEll site in the JH segment (Figure 2). With the exception of one region, bounded by codons 70 through 82A, MLN-a3 was nearly identical (923 of 930 bp) to germline VH7-a3. The region of clustered differences bounded by codons 70 and 82A was striking in that 13 base pairs differed in a 14 codon range, including a codon insertion at position 72. Nucleotide changes in codons 74 to 76 resulted in loss of a Sall restriction site, present in germline VH7-a3. As with l.l-a3 described above, these extensive changes within a small region led us to search known sequences of germline VH genes from the a3 chromosome for segments similar to codons 70 through 82A in MLN-a3. Of the 11 germline VH genes analyzed, one, P289b7 (Currier et al., 1988) had a nucleotide sequence in this region nearly identical to that of MLN-a3 (41 of 42 nucleotides) (Figure 3C). We conclude that a gene such as P26-967 could have

Cell 990

A 1 3 CAG \\\ TCG TTG GAG GAG TCC GGG GGA --GAG (-.A- c--T------_ --___ GAG CA- C-e -T___ --___ -__

V,,l-a3 l.l-a3 v+a3

10 GAC CTG GTC AAG CCT GGG GCA TCC -G- ----c----A- -G- ---G- ___ ___ C-___ -A; -G- --l BamHI

B 55

SO

100

cacagacagtgtgagtgacag\tacctgaccatgtcgtctgtgttttcag -c----g--------------ct------------t---------g--------g-------------c-t-----------+---------g--------g-------------c-t------------t---------g---

V&-a3 l.l-a3 V,,3-a3 V&-a3

C %11 70 SO AAA GGC CGA TTC ACC ATC TCC AAA \\\ ACC TCG TCG ACC ACG GTG ACT CTG CAA ATG ACC --__- --_ _--__ ___ A--G- AGC _-_ AGC CT- -A- --_ --------c--A------------A-- -G- AGC --AGC CTA -A- ------_ ----c--A-

V,,l-a3 MLN-a3 P26-Vbl

D

V,,l-a3 PCRI-2 VH6-a3

ll CTG GTC -----__ ---

AAG CCT GGG GCA -----A- -GC-m --_ -A- -G-

20 TCC CTG ACA CTC ACC TGC ACA GCC TCT GGA TTC TCC TTC -------------A- --------GA- c---__- -__ _-___ --_ -A- ___ ---__ --GA- ---

ATC GCA ---G-----

50 TGC ATT ---------

528 GGT AGT \\\ G-\\\ ---

30 AGT -----

B V,,l-a3 PCR4-2 V,,3-a3

Figure

3. Clustered

Nucleotide

TAT A----

52A GCT A-A--

Changes

AGT

-----

in Diversified

60 GGT AGC ACT TAC TAC GCG AGC TGG GCG AAA GCC __- ----A -GG --__- --_ _--T--T _-------A -GG --------d--T -G-

VH1-a3 and Nucleotide

Sequences

of Potential

Donor

Genes

Diversified VH1-a3 rearrangements are I.+a3 (A and B), MLN-a3 (C), and PCR4-2 (D and E). Donor genes are V&a3 (A), Vn3-a3 and VH4-a3 (B), f26-967 (C), V&e3 (D), and VH3-a3 (E). The nucleotide sequence of VH7-e3 is shown. The locations of the BamHI in l.l-a3 (A) and of the Sal1 in V,l-a3 (C) are indicated by the vertical bars.

donated a nucieotide sequence including codons 70 through 82A to the rearranged VH7-a3 gene in MLN-a3.

those rearrangements that utilized V,7. The rationale for our using size-selected DNA was based on previous Southern analyses indicating that VH7- and JH4-utilizing VDJ rearrangements are on a 4.1 kb Hindiii JH-hybridizing fragment of PBL DNA and that VH7 and J& are preferentially utilized in rabbit VDJ rearrangements (Becker et al., 1990). To ensure that the 4.1 kb Hindiii hybridizing fragment on the Southerns was due to V,7 rearrangements, we repeated the experiment with other restriction

Somatic Diversification of VHl in PCR-Amplified VW Rearrangements in the experiments described above we examined two diversified VDJ rearrangements that utilized VH7. To analyze additional rearrangements, we PCR amplified sizeselected Hindlii-restricted spienic DNA and analyzed

VDJ

--

Figure 4. Restriction Maps of VDJ Genes in Clones MLN-a3 and 41-3 and Germline VH Genes VH1-a3 and V&a3

=

VDJ --

--z

% d I II

vHl-a3

q

-

VH s2--=

= -

0.5 kb

2 d I

a z iz ,

Solid boxes indicate Vn gene segments; open boxes indicate JH gene segments. The restriction map of 41-3 is from Becker et al. (1990) and the maps of germline V~7-e3 and V&-a3 are from Knight and Becker (1990).

Somatic 991

Gene

Conversion

of lmmunoglobulin

12

VDJ Genes

34

’ 1400 -517 -396

75 65

I

Dde

I

Hae

Figure 5. Restriction Fragments MLN-a3 and 41-3 and Germline

111

Hint

I

of VDJ Rearrangements Vn Genes

The 1.5 kb Hindlll-Ncol fragments 5’ of Vn from 41-3 and germline VH genes VH~-a3 and VH4-a3 Ddel, Haelll, and Hinfl. Lanes 1, MLN-a3; lanes a3; lanes 4, V&a3 (see legend to Figure 4 for Hinfl-pUC19 marker are in bp.

in Clones

clones MLN-a3 and were restricted with 2, 41-3; lanes 3, VH1references). Sizes of

enzymes. Southern blots of PBL DNA from an adult a3/a3 rabbit (#203E4), digested with BamHl and EcoRI, were hybridized with a JH probe, PJ5; rearranged JH -hybridizing fragments were found on 6.2 kb BamHl and 3.9 kb EcoRl fragments (Figure 6). As observed previously, a rearranged

-13

RR-

:+.

-4.1 -3.9

-2.0

Figure 6. Southern Slot of Restricted Splenic Homozygous Rabbit Hybridized with Jn Probe

DNA

from

an a%3

Sizes of hybridizing fragments are in kb; fragments containing rearranged JH genes are indicated (R). A weakly hybridizing fragment of approximately 5.6 kb was observed in the EcoRl digest; in subsequent experiments this fragment was not observed and we believe it resulted from partial digestion with EcoRI.

4.1 kb Hindlll fragment hybridized with the JH probe. Assuming that J,+4 was utilized in the VDJ rearrangements, these sizes of BarnHI, EcoRI, and Hindlll fragments were as expected for VDJ rearrangements that utilized VH~ (Becker et al., 1990; Knight and Becker, 1990; K. L. K. and R. S. B., unpublished data). We conclude that the rearranged Jr.,-hybridizing fragment observed in Southern analysis of normal B cell DNA is probably the result of VDJ rearrangements that utilized V,7. This finding gave us the opportunity to use the 4.1 kb Hindlll fragments from normal B lymphocytes to amplify VH7-utilizing VDJ rearrangements. The approximately 4.1 kb Hindlll fragments of splenic DNA from a 4-month-old a3/a3 rabbit (#32E2) were PCR amplified; the 5’primer matched the sequence 206 bp upstream of the V,7 translational start site and the 3’ primer matched sequence from the Jn region. From the amplified DNA, 11 VDJ genes were cloned and sequenced. The regions 5’of the coding region were compared with the region 5’ of the coding sequence of VH7-a3 (Figure 7). Six of the clones appeared to have utilized VH7 as determined by the near identity of their sequences with that of V,7 in the segment 5’ of the coding region. Comparison of the analogous nucleotide sequences of the 11 previously sequenced germline VH genes from the a3 chromosome showed that these 5’ regions differ significantly between various genes and support the idea that this region can be used to assign the utilized gene in the VDJ rearrangement (Figure 6). The other five clones generated by PCR differed significantly from V,7 in the leader intron and in CDRl; they also had a codon insertion at codon 2. These clones either utilized V,7 and underwent extensive diversification, as was observed in clone l.l-a3, or they used a different germline VH gene. Because we could not be certain that these five VDJ rearrangements had utilized VH7, they were not further analyzed in this study. The nucleotide sequences of the six VH7-utilizing VDJ genes differed from the sequence of germline &,I-a3; the majority of the differences occurred in clusters (Figure 7). Two examples of clustered differences are apparent in clone PCR4-2. The nucleotide sequence bordered by codons 15 and 29 (FRl) and by codons 49 and 64 (CDRP) each differed extensively from the germline VH~a3 sequences; moreover, a codon was deleted in CDR2 of PCR4-2. We searched the available sequences of germline VH gene segments from the a3 chromosome for sequences similar to these two regions of diversity. Germ(Knight and Becker, 1990) had a nucleotide line V&a3 sequence at codons 15 to 29 that was nearly identical to (Knight that of PCR4-2 (Figure 3D), and germline l/g-a3 and Becker, 1990) had a nucleotide sequence at codons 49 to 64 that was very similar to the same region in PCR4-2 (Figure 3E). These results indicate that, like the differences in MLN-a3 and l.l-a3 described above, the differences within PCR4-2 could have been generated by a process that utilized germline VH genes as donors of nucleotide sequence. Several other regions of clustered differences exist in the other VDJ genes. Of particular note, the CDRP regions

Cell 992

Figure 7. Nucleotide Amplified V~Wtilizing Splenic DNA

Sequences of VDJ Genes

PCRfrom

Noncoding sequences are shown in lowercase. Amino acid numbers follow that of Kabat et al. (1997). DH and Jr., regions are marked. Dashes designate nucleotides that are identical to those of V,I-e3; slashes represent the absence of nucleotides. Note that the VDJ gene in PCR44 is a nonfunctional rearrangement as VH7 and the JH5 gene segments are not in the same reading frame.

______--- -__ __-_-- ______--_ ____-_ --_ _.- ---

___ -c-G-

___

_-___ -__ _.- __- _-___ _-_ _-_ __- __- c-- -c____-______ _-- --- --_ TTG TTG TTC TCT TTC TTC

XC TGC TCC CCG TCC TGC

of clones PCR4-4 and PCR4-16 differ extensively from the analogous germline VH7 segment; besides nucleotide changes, each shows the loss of a codon. In PCR4-4, this region is similar but not identical to the diversified CDR2 region of PCR4-2. In PCR4-16, this region is distinct from the CDR2 of all other PCR clones. A search of the 11 available germline VH genes on the a3 chromosome did not identify a gene with a similar CDRP region. DH Segments within the VW Gene We identified the DH segments of the VDJ genes by comparing nucleotide sequences of the VDJ genes with those

of germline V,.,7-a3 and with germline JH gene segments. Those segments in the VH-D”-JH joining region not apparently contributed by the VH7 or JH germline genes were considered DH segments (Figures 2 and 7). Comparison of the nucleotide sequences of these DH segments with those of the known germline DH gene segments (Becker et al., 1969) did not reveal significant sequence similarity except for a short region (TGClGGTTGT) in MLN-a3 that has homology to the D2a and D2b germline sequences (Becker et al., 1969). To localize the germline counterparts of the other DH regions, we synthesized oligomer probes for the DH segments in clones

Somatic 993

Gene

Conversion

of lmmunoglobulin

VDJ Genes

V,,l-a3 V,,4-a3 V,,34

vu25 V,,l-a3 V,,4-a3 "I,34 5

caaatagggcctccctctgcccatgaaaaccagcccagccctcaccctgcagctctggcacaggagctccagccccagca ----------\-------------------------------------------------------------------ga-g-----------------------------------------------------------------------------

“142 “J

------------\-------------------~_--_--_------__~---_~~~~~~~~~~~_--_--_------------------------

VW4 V,,2-a3 V,,3-a3 V,,5-a3 V,,6-a3 V,,l-a3 V&a3 v,,4-a3 Vu34

ctcccaggtgtccactcagtgatcgcactcaacacagaca=t=~~=

---\\\\\\\\\\\\\\-------~----g---------~------

vu25 w

---------------------------------------g-----__--_----__-------_----c-----~-------_________________-_____________________g~~~~~~ -----------------tc-c------------------9------

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-19 ATG --___ ---

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CTG --__---

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CTT _-___--

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",,I-,i3 v,,4-d3 ",,34 5

w VI,2

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agaCaC~gaCagtgtgagtgaCag\tacctgaccatgtcgtctgtgttttcag ---------g-------------c-t-----------t---------g---a-------g--------------c-t--------9-a-----------g-t-----g--------------tgc---------g-~-----------g-t---------g-----c--------tgt---------g-=-----------g-t---------g----------g--tgt----------------------g-------g-a--------tgt-ac--agaga-ga-gga--cc-=~~==t=~ ---------g-------------c-t------------t---------g-----------g-------------tgtg-------g-a------------g-----------g-------------tgtg-------g-a------------g-----------,\-------------tgtg--------g-~-----------g---

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VI,4 '+,2-a) V,,3-a3 V,,5-a3 v,+a3 V,,l-a3

Figure

"I

IIII___---___-~TTI~-______IIIIII'I

8. Nucleotide

Sequence

Comparison

of Regions

5’ of Codon

GT -_ __ ----

-3 GTC -__ -__ ___ -__ _-_

__ --_ __

___ ___ ___ ___

1 of 11 Germline

Noncoding sequences are shown in lowercase. Leader sequence -19 to -3 is shown (Bernstein et al., 1985) and VH7-a3 through VJ-a3 (Knight and Becker, 1990). Dashes the absence of nucleotides.

PCFH-1, PCR4-2, PCR4-3, and PCW-16 and probed Southern blots of the cosmid clones that contained the complete DH region from an a3 chromosome (Becker et al., 1989). These DH segment probes did not hybridize to the cosmid clones containing the germline DH region from the a3 chromosome (data not shown). From these data we conclude that the DH segments in the VDJ rearrangements do not derive from germline DNA between the VHl and Jnl genes. Discussion Diversification of V,+l Genes After comparing nucleotide sequences of rabbit VDJ genes with their germline counterpart, VH1-83, we conclude that these genes were extensively diversified by gene conversion events. The validity of this interpretation is dependent on identification of the diversified gene as VHl-a3. Several lines of evidence support the idea that the genes we analyzed were VDJ rearrangements of VHF a3. First, Southern blot analysis of PBL DNA showed that

VH Genes

from the 8s Chromosome

in uppercase. Sequences are from VH2, V#, V~25, and V~34 designate nucleotides identical to V,7-a3; slashes represent

normal B lymphocytes preferentially utilize VH gene(s) in VDJ rearrangements that consistently resolve in the same-sized restriction fragments; the sizes of the Hindlll, BamHI, and EcoRl fragments with the rearranged genes, 4.1 kb, 6.2 kb, and 3.9 kb, respectively, were those expected if VHl-a3 had been used in the VDJ rearrangements. Second, the VDJ gene l.l-a3 was cloned in phage and the 7 kb of DNA upstream of the VDJ gene showed a restriction map identical to that of the region upstream of germline VHl. Furthermore, the sequence of the VH gene upstream of the VDJ rearrangement, l.l-a3, in phl.1 was identical to that of germline VH2, as expected if V,.,la3 had been used in the VDJ rearrangement. The other VDJ gene cloned in phage, MLN-a3, also resided on a 4.1 kb Hindlll fragment and its restriction sites upstream of the VDJ gene rearrangement were identical to those upstream of V,l-a3. In addition, the 515 base pairs of sequence 5’ of the VDJ were identical to the analogous region 5’ of germline VHl. In our view, these data unequivocally demonstrate that 1.1-a3 and MLN-a3 utilized V,l in their VDJ rearrangements.

Cell 994

Evidence that the PCR-amplified VDJ rearrangements derived from VH7-a3 comes from two facts: all of the genes were amplified from approximately 4.1 kb Hindlll fragments of spleen DNA (the sized fragment containing rearranged VH1 genes), and the promoter, leader, and intron nucleotide sequences were nearly identical to that of germline VH7-a3. Although we cannot rule out the possibility that other VH genes have identical promoter, leader, and intron sequences, we consider this unlikely. We must consider the possibility that genes we designated as donor genes for gene conversion events are, instead, the utilized VH gene. For example, P26-9b7 rather than V~7-a3 could be the utilized gene for MLN-a3; however, MLN-a3 and f26-967 differ by 33 nucleotides throughout the VH region, making this possibility unlikely (MLNa3 and VH7-a3 differ by 19 nucleotides in the Vu region). Similarly, it is unlikely that any of the other designated “donor” Vu genes were, instead, the “utilized” VH gene. Considering all the data, we are confident that all the VDJ genes we analyzed in this study utilized V,+7-a3. Generation of Antlbody Diversity by Somatic Gene Conversion Diversification of the V regions in rabbit VDJ rearrangements is striking in that the observed variations occur primarily as clusters of nucleotide changes and codon insertions/deletions. In mice, most somatic diversification of VDJ genes appears to occur by point mutations because the variations occur as scattered single nucleotide changes (Kim et al., 1981; Gearhart et al., 1981; Pech et al., 1981; Krawinkel et al., 1983; Chien et al., 1988; Griffiths et al., 1984; Clarke et al., 1985; Cumano and Rajewsky, 1988; Rajewsky, 1989; Wysocki and Gefter, 1989; Shlomchik et al., 1990). It is unlikely that the clustered diversity regions in rabbit V,.,7 rearrangements result from random somatic mutation. The fact that we identified, for five clustered diversity regions, sequences similar or identical to upstream germline Vu genes, strongly suggests that upstream Vu genes are involved in diversification of V,7 rearrangements. Similar observations have been made in chickens, which utilize only a single VH and a single VI gene segment in their lg gene rearrangements (Reynaud et al., 1989). In this species, antibody diversity is generated by gene conversion, whereby V pseudogenes 5’ of the functional V gene donate nucleotide sequences to the variable regions of the VDJ gene rearrangement. An important prerequisite for these gene conversion events in chickens is that the donor and recipient genes have a high degree of sequence similarity upstream of the region of conversion (McCormack and Thompson, 1990); such a region of sequence similarity is particularly apparent in rabbit VH genes of clones 1.1.a3 and MLN-a3 and their potential donor genes, V&a3 and P26-967, respectively. We suggest that rabbit VH genes are diversified in part by gene conversion with Vu genes 5’ of V,7 acting as donor sequences. However, we cannot rule out the possibility that the appearance of donor sequences is due to double recombination events or to another unknown mechanism. In the case of double recombination, both the donor and recipient genes would be modified. Although modification

of the donor gene does not seem to occur during chicken VDJ diversification (Carlson et al., 1990), we currently cannot evaluate this possibility in rabbit B lymphocytes. Gene conversion as a mechanism for generating antibody diversity in mammals has been extensively investigated, but there is only limited evidence that such conversion events occur in somatic cells (Krawinkel et al., 1983; Cumano and Rajewsky, 1986). However, considerable evidence exists indicating that gene conversion events have occurred in mammalian germ cells. Slightom et al. (1980) first obtained evidence for germ cell gene conversion in mammals when they were studying human fetal globin genes; subsequently, many examples of gene transfer in germ cells have been found for murine major histocompatibility complex (reviewed in Geliebter and Nathenson, 1987) and immunoglobulin variable and constant region genes (reviewed in Wysocki and Gefter, 1989). Thus, gene conversion as a means of generating variant genes can occur in mammals, especially in mice, but, in contrast to rabbit, this mechanism does not seem to operate extensively in B lymphocytes to generate antibody diversity. For three of the five examples of gene conversion in rabbit VDJ genes (Figure 3) the potential donor VH gene that we identified differed from the converted rearranged VH gene by one or a few nucleotides. There are several possible explanations for the discrepant sequences. First, the actual donor VH gene may not have been the one we identified but was instead another very similar VH gene for which the sequence is not yet determined. We consider this a likely explanation for at least some of the nucleotide variation between the donor and ‘converted” rearranged VH genes since the data bank we screened for potential donor genes had sequences for only 11 out of the few hundred, total, germline VH genes. Second, the nucleotide differences between the donor and rearranged VH genes could be due to the rearranged VH gene having undergone more than one gene conversion event with different donor genes, as was shown to occur in chicken light chain genes (Reynaud et al., 1987). Still another explanation would be that these nucleotide differences between the potential donor and the converted rearranged gene arose by somatic point mutations of the latter. Finally, in the PCR-amplified clones, the nucleotide differences could represent errors introduced during PCR amplification; we consider this possibility unlikely, as the nucleotide differences between donor and rearranged genes were generally not those typically introduced by Taq polymerase, that is, A-T to G-C transitions (Keohavong and Thilly, 1989). Although many of the nucleotide differences between VDJ genes and germline sequences are clustered, some changes appear to be single base mutations, for example in codons 23 and 32 of MLN-a3 (Figure 2). These isolated base pair differences may result from somatic point mutations or they may result from a gene conversion event between two genes that are nearly identical. Within the coding regions of the eight VDJ genes that were analyzed, a total of 104 base pairs were found to be different from the nucleotide sequence of germline VH7-a3; nucleotides for

Somatic 995

Gene

Conversion

of lmmunoglobulin

VDJ Genes

78 of these 104 changes (75%) can be found in the 11 germline Vu gene segments already sequenced (Bernstein et al., 1985; Currier et al., 1988; Knight and Becker, 1990). It is likely that many of the remaining nucleotide differences will be found as the sequences of other germline VH genes are determined. We believe, however, that some of the diversification of these genes may also be the result of random somatic mutation. The gene conversion events were found in the leader intron, FRl, CDRl, CDR2, and FR3 of the rabbit VDJ genes. It has long been established that the diversification of CDR regions affects antigen recognition, and hence diversification of these regions is expected to increase the total repertoire of antigen binding molecules. FRl and FR3, on the other hand, are not typically thought of as regions important for antigen recognition. Capra and Kehoe (1974), however, previously identified a portion of FR3 as a hypervariable region in human antibodies. Recently, Weigert and colleagues (Shlomchik et al., 1990) have shown amino acid changes in this region to correspond with the high affinity murine autoimmune antibodies to DNA. It seems likely that this portion of thevariable region could be involved in antibody-antigen interactions based on its being in the p-loop that is immediately adjacent to the classical antigen binding site. Thus, in some antibodies, this part of FR3 may have a role in antibody-antigen interactions either by direct contact with the antigen, or by affecting the tertiary structure of the antigen binding site. D Region Diversity We have shown here and in previous work (DiPietro and Knight, 1990) that D region (CDR3) diversity is extensive in VDJ rearrangements and that the D regions in VDJ rearrangements from splenocytes of adult rabbits bear little resemblance to germline Dl, D2, or D3 gene segments (Becker et al., 1989; R. S. B. and K. L. K., unpublished data). Thus either the germline D segments Dl, D2, and D3 are not used in these VDJ rearrangements, or they are used but then undergo extensive alterations and/or mutation. Because D regions of VDJ rearrangements from newborn spleen (R. S. B. and K. L. K., unpublished data) and from leukemic B cells of young rabbits (Becker et al., 1990) are germline in nature, we believe that germline Dl, D2, and D3 gene segments are used in VDJ rearrangements in adult B lymphocytes. Hence, we suggest that extensive diversification of D regions occurs after VDJ rearrangements. Similar observations were made in chicken B lymphocytes (Reynaud et al., 1989) where it seems that D regions are extensively diversified after VDJ rearrangements; this diversification occurs by gene conversion with regions 3’ of the VH pseudogenes acting as donor sequences. We performed a limited analysis of regions 3’ of rabbit germline Vu genes for regions with nucleotide sequences similar to diversified D regions. So far, none have been identified. Although we do not understand the mechanisms that diversify the D segments, there is evidence that random point mutation is not the primary mechanism. The diversified D segments in some of the PCR-derived VDJ clones contain tandemly repeated nucleotide se-

quences; for example, PCRC1 has the sequence TGGTAG repeated three times. These data suggest the diversification process may involve mechanisms that duplicate short regions of DNA. The rabbit has provided us a unique opportunity to analyze somatic diversification of VDJ genes in mammalian B cells. Mice utilize many of their several hundred germline VH genes in VDJ rearrangements, which makes it difficult to assign the exact germline counterpart of these diversified genes. On the other hand, rabbits preferentially utilize one VH gene in their VDJ rearrangements, which makes identification of the germline counterpart quite straightforward. Comparison of the diversified rabbit VDJ genes with their germline homologs indicated that diversification occurred by somatic gene conversion. Of the seven rabbit VDJ genes examined, we identified at least five potential gene conversion events. The high frequency of these events suggests that gene conversion is a major means of generating antibody diversity in rabbits. Our data provide clear evidence for somatic gene conversion of VH genes in a mammalian species. Experimental

Procedures

Germllne DNA Genomic DNA was prepared (Blin and Stafford, MLN. and PBL of #/a3 homozygous rabbits, heavy (Dray et al., 1974) maintained at Loyola University type of the rabbits was defined by pedigree and

1976) from spleen, chain haplotype G Chicago. The genoserologic analysis.

Cloning of VDJ Rearrangements A partial Mbol library was prepared in phage EMBL4 vector with DNA from spleen and MLN of a 6-month-old a3/a3 rabbit, #207A3. The lie brary was screened with a rabbit Jn probe, PJ5 (Becker et al., 1989) and with the pan-Vu probe, ~161 (Gallarda et al., 1985). One clone, phl.1. hybridized with both probes and was isolated and restriction mapped. Hindlll fragments of approximately 3.5-4.5 kb were isolated from MLN DNA of a 6-month-old a%s3 rabbit (#184B5) and were cloned into phage Charon 28. The phage library was screened with the Jn probe, PJ5 (Becker et al., 1989) and one hybridizing clone, MLN-a3, was isolated. The size of the Hindlll insert of MLN-a3 was 4.1 kb; this fragment was cloned into pUCl8 and restriction mapped. PCR amplification of VDJ genes was performed with Hindlll fragments of approximately 3.8-4.3 kb ikrolated from splenic DNA of a 4-month-old a3h3 rabbit (#32E2). The nucleotkle sequences of primers used for PCR amplification were S’GTCTAGAATCCTATCACAGCCATCACTCTACAT-3’ from the promoter region of the V,J gene (206 bp 5’ of the translational start site) and WXCGAGAATTCTGAGGAGACGGTGACCAGGGTGCC-3’ from germline Jn region (Becker et al., 1989). The amplified VDJ genes were cloned into pUC18, and 11 VDJ containIng clones were identified by hybridization with the Jn probe, PJ5. Nucleotlde Sequence Analysis Restriction fragments containing the VDJ genes from the phage clones were subcloned into Ml3mpW19 (Yanisch-Perron et al., 1985). and the nucleotide sequence, in both orientations, was determined by the dideoxy chain termination method (Sanger et al., 1977) with the Sequenase kit supplied with Taq polymerase (United States Biochemical Corp., Cleveland, OH). Double-stranded nucleotide sequencing was performed on both DNA strands to determine the nucleotide sequence of PC&lified VDJ genes. The 11 germline Vn genes from the a3 chromosome used in the computer analysis for donor sequences were V,2-a3 through V$a3 (Knight and Becker, 1990) Vd4 and V,25(Bernstein et al., 1985) and P26.9a3, P26.9b1, and f26.9~2 (Currier et al., 1986). Southern Southern

Analysis blot analysis

of restricted

PBL genomic

DNA from the a%3

Cell 996

homozygous rabbit (#203E4) was performed with the Jr., region probe, PJ5 (Becker et al., 1989) as previously described (Southern, 1975). The D region oligomers were synthesized by the Macromolecular Center at Loyola University Chicago, Medical Center and were as follows: PCRclDn, 5’-AGATTTGGTAGTGGTAGTGGTAGTGGTGTCTACTGG-3’; PCR4-2D+,, 5’TAATGGTGCTAGTGGTGCT-3’; PCR4-3Dn, 5’-ATAGGGTCTACTAATGATGGTTAT-3’; and PCR4-16Dn, 5’-AAGGATGGTATTGGTAGTGCTTAT-3’. The Southern blots were hybridized with kinased oligomers at 42% in hybridization buffer containing 6x sodium chloride-sodium citrate and were washed at 50% in washing buffer containing 2x sodium chloride-sodium citrate (Sambrook et al., 1989).

We gratefully acknowledge the expert technical assistance of Mr. ShiKang Zhai and Ms. Jessy Thomas. We acknowledge contributions of A. Golden and Dr. M. Patino to the early phases of experiments with clones phl.l and MLN-a3, respectively. We also appreciate helpful discussions of the manuscript with Drs. Latham Claflin and W. Carey Hanly. This research was supported by grants from the Public Health Service, National Institutes of Health, Al16611, from the Cancer Research Foundation of Chicago, and from Biomedical Research Support Grant Program of LUMC. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. August

of rabbit

Gatlarda, J. L., Gleason, K. S., and Knight, K. L. (1985). Organization of rabbit immunoglobulin genes. I. Structure and multiplicity of germline Vn genes. J. lmmunol. 135, 4222-4228. Gearhart, P J., Johnson, N. D., Douglas, R., and Hood, L. (1981). IgG antibodies to phosphorylcholine exhibit more diversity than their IgM counterparts. Nature 297, 29-34. Geliebter, concerted

J., and Nathenson, S. G. (1987). Recombination and the evolution of the murine MHC. Trends Genet. 3, 107-112.

Griffith.% G. M., Berek, C., Kaartinen, M., and Milstein, C. (1984). Somatic mutation and the maturation of immune response to P-phenyl oxazolone. Nature 312, 271-275.

Acknowledgments

Received

Dray, S., Kim, 8. S., and Gilman-Sachs, A. (1974). Allogroups lg heavy chains. Ann. Inst. Pasteur 125C, 41-47.

6, 1990; revised

September

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VDJ Genes

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Accession

The accession numbers M55381 to M55388.

Number for the sequence

reported

in this paper

are

Somatic diversification of immunoglobulin heavy chain VDJ genes: evidence for somatic gene conversion in rabbits.

Rabbits preferentially utilize only one of their multiple functional germline immunoglobulin VH genes. This preferential usage of one gene, VH1, raise...
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