Cell, Vol. 60, 963-970,

March

23, 1990, Copyright

0

1990 by Cell Press

Molecular Basis of the Allelic Inheritance of Rabbit lmmunoglobulin VH Allotypes: Impkations for the Generation of Antibody Diversity Katherine L. Knight and Robert S. Becker Department of Microbiology Stritch School of Medicine Loyola University Chicago Maywood, Illinois 60153

Summary Rabbits ate unique in that their immunogiobulin VH regions bear aliotypic markers encoded by allelic genes. The presence of these markrs on most serum immunoglobulins is difficult to explain, as the germline contains several hundred VH genea We cloned VH genes tim normal rabbits of the VHa aliotypes al, a2, and a3 and from a mutant a2 rabbit, Alicia, which expresses almost no a2 allotype. The D-proximal VH gene WHl of normal rabbits encoded prototype al, a2, or a3 allotype VH regions in al, a2, or a3 rabbits, respectively; VHl was shown to be preferentially utilized in leukemic rabbit B cells. This VH7 gene was deleted from the germline of the Alicia rabbit. These data suggest that the allelic inheritance of a allotypes results from preferential utilixation of V/f1 in VW rearrangements. We suggest that antibody diversity in rabbit primarily results from somatic hypermutation and gene conversion. introduction Three VH region allotypes, al, a2, and a3, of rabbit immunoglobulins were discovered over 30 years ago (Oudin, 1956a, 1956b; Dray and Young, 1958; Dubiski et al., 1959) and were shown to be inherited in a simple Mendelian fashion. These VH region markers, unique to the rabbit, were instrumental in understanding immunoglobulin gene organization and expression, especially the phenomena of allelic exclusion (Pernis et al., 1965; Cebra et al., 1966), linked inheritance of VH and CH genes, and cis expression of VH and CH genes (Kindt et al., 1970; Landucci-Tosi et al., 1970). The observation that the a allotypic specificities are found on heavy chains of several immunoglobulin isotypes (Todd, 1963; Kindt and Todd, 1969) challenged the one gene-one polypeptide chain dogma and led to the development of “new” theories of antibody formation (Dreyer and Bennett, 1965; Lennox and Cohn, 1967). The allelic inheritance pattern of rabbit VHa allotypes presents an enigma for immunologists and geneticists. Eighty to ninety percent of serum immunoglobulin molecules bear the nominal VHa allotypic specificity (Dray et al., 1963b), yet these same molecules have diverse antigen binding specificities, implying diverse VH regions. The VHa allotypic specificities correlate with particular amino acids at several residue positions distributed within the VH framework regions (FRs) 1 and 3 (Mage et al., 1984), whereas the antigen binding specificities contributed by the heavy chain reflect amino acid sequence variations in the complementarity determining regions

(CDRs). The rabbit genome contains hundreds of VH genes (Gallarda et al., 1985; Currier et al., 1988), and we have generally assumed that most of the genes are used to generate antibody diversity. However, if this is true, it seems to follow that there must be a mechanism to maintain the allotype-encoding regions of the multiple VH genes on a single chromosome, for otherwise, meiotic recombinations among the VH genes would shuffle the VHa genes such that the allelic behavior would be lost after several generations of progeny. Several possibilities have been put forward to explain the allelic inheritance of these allotypes (reviewed in Mage, 1981; Kindt and Capra, 1984). One possibility is simply that crossovers among the VH genes are, by some unknown mechanism, suppressed. Alternatively, crossovers may readily occur among the VH genes, resulting in a similar VH repertoire among rabbits of different VHa allotypes; in this case, the “allelism” of VHa genes could be due to an unidentified allelic regulatory gene (Strosberg, 1977). Another possibility is that the 80%-90% of immunoglobulin molecules that have the VHa allotypic marker are derived from one, or a small number of, closely linked VH gene(s) rather than from the several hundred VH genes in the germline (Currier et al., 1988). Support for restricted utilization of germline VH genes in functional VDJ rearrangements has recently been obtained by analysis of cDNA clones from splenic poly(A)+ RNA (DiPietro and Knight, 1990). To understand the allelic inheritance pattern of the allotypes al, a2, and a3, we began to compare the VH gene repertoire of al, a2, and a3 rabbits as well as of a mutant a2 rabbit, Alicia, that expresses low levels of a2 immunoglobulin and yet has normal immunoglobulin levels (Kelus and Weiss, 1986). We describe here studies of the allelic 3’-most VH germline genes in al, a2, a3, and Alicia rabbits. Based on observations from these studies and from analysis of VDJ genes from leukemic B cells, we propose that the allelic inheritance pattern of the a allotypes is due to preferential utilization of the 3’-most VH gene VH7. Such preferential utilization of one VH gene would seemingly limit the extent of antibody diversity in rabbit immunoglobulin. We propose that the majority of antibody diversity is generated by a combination of mechanisms that could include somatic hypermutation and gene conversion. Results Aiielic VHl Genes The 3’-most VH genes (D-proximal) of homozygous alla’, a2/$, and a3/a3 rabbits were cloned from cosmid libraries. The libraries were constructed in the pTL5 vector, from liver or sperm DNA, and were probed with a single copy D region probe (probe A; Becker et al., 1989). The clones that hybridized with probe A were hybridized with a panVH probe, ~181 (Gallarda et al., 1985), and pOSitiVe clones were selected, grown, and restriction mapped. VH genes

Figure 1. Restriction Maps of Cosmid Clones Containing the 3’-most VH Genes from Homozygous alla’, azia2, and a3la3 Rabbits Homozygous rabbits were al/a’ (A: Cos 2.1al). az/a2 (8: Cos 8-a2), and a3/a3 (C, Cos 9.2-a3). The region of each clone that hybridized with probe A is indicated. Solid boxes represent VH genes that hybridized with the panVH probe ~181. The sizes below the restriction maps are in kb. The 5’to Yorientation of some of the VH genes is indicated by arrows.

I 0

I 20

10

cm

B

-w

I

I 30

I

a-a2

w

VH4

SVH3

u

llVH2

VHl

u

u

u

u Probe II

Xba I sac

II

I

III I

BamH I Hind Ill EcoR

I I

I

I

I

I I

I

I

I

I

III

III I

I,

1

/

I

I

I I

I

I

II

I

I

I

I

I

I

I

I

Sal I

I

Kpn I spe

A I I

II

1

I

I

I

I

Sfi I

I

I

I

I

t

,

10

0

I

I

20

30

Cos %2-a3

C -

w

SVH7

VHB

SVH5

VH4

w VH3

4VH2

W VHl

I

I

I

I

1

I

u Pro&

Xba I sac

1

II

I

BamH I

I

I

I

I

I

I

I

Hind III EcoR

I

I

I

I

I

I

I I

III

I

I

II I

I

I

II

I I

I

Sal I Kpn I

I

I

II

I

A I

I

I

, I

I

Cla I MIU I

were localized by Southern analysis (Figure 1). The VH gene-containing cosmid clones from the al, a2, and a3 rabbits each had a unique restriction map, indicating that the al, a2, and a3 chromosomes were distinct. The cosmid clones contained from four to seven VH genes, sepa-

I

rated by 3-7 kb of DNA; the VH genes in the cosmid clones were designated VHl-VH7; with VHl being the 3’-most VH gene. We determined the nucleotide sequences of VU7 genes from the al, a2, and a3 chromosomes. All three genes ap-

A$lic

Inheritance

of lmmunoglobulin

VH Allotypes

Figure 2. Comparison of Translated Amino Acid Sequence of V/-/7-a7 and VH4-el with the Partial Amino Acid Sequence of Pooled al Immunoglobulin

amer and heptamer sequences 3’of the VH genes are underlined. Amino acid numbering ered as: FRl, amino acids l-30; FR2, amino acids 3649; FR3, amino acids 66-94.

peared to be functional, as they each encoded a typical VH region with a 19 amino acid leader peptide and had the conserved RNA splice sequences at the intron-exon boundaries and conserved recombination signal sequences 3’ to the VH-encoding region (Figures 2, 3, and 4). The translated sequences of the VHl genes were compared with one another (Figure 5) and since allotype-associated amino acids reside in the FRs of VH, the translated VH7 sequences were compared with the partial amino acid sequences of FRs of pooled immunoglobulin of known VHa allotype (Figures 2, 3, and 4). The VH region encoded by VH7 from the al chromosome, VHSal, was identical to the sequence found for the VH FRs of pooled al serum IgG from normal, unimmunized rabbits (Mole et al., 1971; Johnstone and Mole, 1977; Kabat et al., 1987). Also, VH7-a7 encoded each of the four ascribed al allotypeassociated amino acids at positions 10, 13, 84, and 85 (Figure 2; Mage et al., 1984). Thus, it appears that VH7-al, from the al chromosome, encodes “prototype” al molecules. Similarly, VH7 from the a2 chromosome, VH7-a2, encoded VH FRs identical to the partial amino acid sequences of FRs of VH of pooled a2 serum IgG (Mole, 1975; Johnstone and Mole, 1977) and it encoded 10 of the 11 predicted a2 allotype-associated amino acids at positions 5, 8, 12, 18, 17, 85, 57, 70, 71, 74, and 75 (Figure 3; Mage

The partial amino acid sequence of pooled al immunglobulin was from Kabat et al. (1967). The al allotype-associated amino acid residues (Mage et al., 1964) are marked by asterisks. The nucleotide sequences of the intron between the first (leader peptide) exon and the VH exon segment are shown in lowercase Iettens. Dashes represent identity; the pound sign (#) represents more than three amino acids found in pooled al immunoglobulin; gaps within the pool-al sequence (residues 53-57) indicate residues that were not identified in the protein sequence (Kabat et al., 1967); slashes (\) denote absence of nucleotide. Conserved nonis from Kabat et al. (1967); framework regions are consid-

et al., 1984). Thus, it appears that VH7-a2, from the a2 chromosome, encodes “prototype” a2 molecules. VH7 from the a3 chromosome, VH7-a3, appears to encode an a3like molecule, as its FR sequences (Figure 4) were nearly identical (52 of 54 residues) to the partial amino acid sequence found for the FRs of VH of pooled a3 serum IgG (Mole et al., 1971; Mole, 1975; Johnstone and Mole, 1977). This gene also encodes the one potential a3 allotypeassociated amino acid Asp at position 10 (Mage et al., 1984). The finding that VH7 from the al, a2, or a3 chromosome encodes “prototype” al, a2, or a3 VH regions, respectively, confirms the serologic and genetic data showing that the VHa allotypes are encoded by allelic genes. These data also suggest that this gene and/or one(s) very similar to it is used in S cell VDJ rearrangements. Analysis of Germline W/2-VH7 Genes The VH4 genes from all three chromosomes, along with VH3 and VH6 from the a3 chromosome, appear to be functional and encode VH regions similar to those encoded by VH7 from the same animal (Figures 2, 3, and 4). In all cases, however, the amino acid sequences encoded by the VH4 genes are less similar to the amino acid sequences of pooled normal IgG than are the sequences en-

Figure 3. Comparison of Translated Amino Acid Sequence of VHGa2 and VH4-a2 Genes with the Partial Amino Acid Sequence of Pooled a2 lmmunoglobulin Gaps within the pool-a2 sequence represent residues that were not identified in the protein sequence (Kabat et al., 1967). The a2 allotypeassociated amino acid residues (Mage et al., 1964) are marked by asterisks; residues 64 and 65 are not included as a2 ailotype-associated residues. Allotype-associated residues that were not identified in the pool-a2 sequence (Mole, 1975; Johnstone and Mote, 1977) are designated by parentheses. See the legend to Figure 2 for further details.

Cdl 966

coded by the VHl genes. VHl-al encodes FRs identical to the VH FRs of pooled al IgG sequence, whereas VH4-al encodes FRs that differ from those of pooled al IgG by 8 amino acids (Figure 2). VH4-a7 does, however, encode the four ascribed al allotype-associated residues (Mage et al., 1984). VH4 of the a2 chromosome, VH4-a2, encodes FRs that differ from those of pooled a2 IgG by 3 amino acids but has 7 of the 11 ascribed a2 allotype-associated residues; VHPa2 encodes FRs identical to those of pooled a2 IgG. VH3-a3, VH4-a3, and VH&a3 encode FRs that differ from those of pooled a3 IgG sequences by 8, 4, and 10 amino acids, respectively, whereas VHl-a3 encodes FRs that differ by only 2 amino acids from those of the pooled a3 IgG (Kabat et al., 1987). Thus, although functional VH4 genes (plus VH3 and VH6 of a3 rabbits) appear to encode VH regions similar to the prototypic al, a2, or a3 allotype molecules, the VH7 gene, on each chromosome, encodes molecules most similar to those found in normal serum immunoglobulin, indicating that the majority of serum immunoglobulin may be en-

Figure 5. Comparison of the Translated VHl-al, VHl-a2, and VHl-a3

Amino

Acid

Sequences

Allotype-associated residues for al and a2 VH regions are circled. Amino acids are numbered according to Kabat et al. (1937).

of

coded by VHl rather than by VH4 (or VH3 and VH6 in a3 rabbits). The nucleotide sequences of the remainder of the cosmid VH genes, i.e., VH2 and VH3 from the al and a2 chromosomes and VH2, VH5, and VH7 from the a3 chromosome, showed that these genes are pseudogenes as a result of stop codons or truncations in the VH gene segments. With the exception of VH3-a3 (see below), the sequences for these pseudogenes are not presented herein but are deposited in GenBank. Preferential Utilization of VH7 in Leukemic B Cells Eu-myc transgenic rabbits develop polyclonal B cell leukemias (Knight et al., 1988) and we cloned eight VDJ gene rearrangements from leukemic cells of three such transgenie rabbits. The nucleotide sequences of the eight rearranged VDJ genes were determined (Becker et al., 1990) and compared with the germline VH sequences reported above. These comparisons show that seven of the eight clones had sequences identical to that of VHI-al, VHI-a2, or VHCa3 (see Figures 2-4; see also Figures 3-5 in Becker et al., 1990). These data suggest that VH7 was used in VDJ rearrangements in these leukemic B cells. To determine whether VH7 was indeed the utilized gene, the VDJ gene from a leukemic B cell line, PBL-1, was cloned from a cosmid library, and the DNA 5’ of the VDJ gene was analyzed for restriction sites and for VH genes characteristic of VH2, VH3, and VH4 (Figure 8). Since the VDJ gene of PBL-1 appears to be on the a2 chromosome (Becker et al., 1990) the restriction map of the PBL-1 cosmid Cos 14.1-a2 was compared with the restriction map of the cosmid Cos 8-a2, representing the germline a2 chromosome (Figure 1). The restriction sites 5’ of the germline VH7 (Cos 8-a2) and 5’ of the VDJ gene of PBL-1 (Cos 14.1-a2) were identical, as were the locations of VH2, VH3, and VH4. These data confirm that VHl had been utilized in the VDJ rearrangement in PBL-1.

;ililic

Inheritance

of lmmunoglobulin

W-l Allotypes

Figure 6. Restriction Map of Cosmid Clone Cos 14.1~a2 Containing the VW Gene from the a2 Chromosome of PBL-1 Cell Line The regions that hybridized with the pan-VH probe, Cu, heavy chain enhancer (Ep), and switch region (SW) probes are indicated. The sizes below the restriction maps are in kb.

0

10

20

30

Deletion of Germline V/f1 Gene Correlates with Loss of VHa Allotype Expression Alicia rabbits, homozygous for the mutant a2 heavy chain chromosome ah, express low levels of a2 immunoglobulin; the low level of a2 immunoglobulin is compensated by an increase of socalled a-negative molecules. The genetic basis of the defect is not known, but genomic blot analysis of a/i DNA indicated that no major deletions had occurred within the VH chromosomal region of the a/i chromosome (Kelus and Weiss, 1988). Based on the results presented above, we reasoned that because VU7 is preferentially utilized and encodes a2 molecules in normal a2 rabbits, the mutation in a/i may have altered or deleted V/-/7. As a consequence, little or no a2 immunoglobulin would be synthesized by these Alicia rabbits. To test this hypothesis, a cosmid library was constructed from spleen DNA of a homozygous a/i/a/i rabbit, and clones containing the 3’-most VH genes were identified with probe A. One clone, Cos 29.3-Ali, was restriction mapped, and the VH genes were localized by Southern analysis with the pan-VH probe ~181 (Figure 7). Comparison of the restriction map of this clone with the restriction map of Cos 8-a2, containing V/-/7-VH4 from the a2 chromosome of a normal a2 rabbit, revealed that

Cos

VHS

VH7

VH6

I

I

I

the DNA from the Alicia rabbit was missing VH7 and VH2 (Figure 8). The restriction sites 5’ of VH2 and 3’ of VH7 in Cos 8-a (normal a2 chromosome) were found in the Cos 29.3-Ali, but no restriction sites corresponding to those of VH7 and VH2 were present in the Cos 2934li clone. Thus, the a/i DNA has a deletion within its heavy chain chromosomal region. The region deleted is 10 kb in length, as can be observed in the size of the Sfil fragment; this fragment is 23 kb in Cos 8-a2 (normal a2 chromosome) and 13 kb in Cos 29.3~Ali (Figure 8). To rule out the possibility that the deletion observed in Cos 29.3~Ali was due to a cloning artifact, we partially mapped an overlapping cosmid clone, Cos 18.1~Ali (Figure 8). The Sfil fragment of this clone was identical in size to that of Cos 29.3-Ali (13 kb), indicating that the decreased size of the Sfil fragment in clone Cos 29.3-Ali was not the result of a cloning artifact. That a/i differs from its parental a2 chromosome in an Sfil restriction site in the VH-DH region has been recently demonstrated by R. Mage and her colleagues(NIH; personal communication). The restriction map of the Ali cosmid clone indicated that the 3’-most VH gene of ali was V/+3. The nucleotide sequence of VH3-ali was identical to the sequence of VH3 from a normal a2 chromosome (Figure 9) and appeared

Figure 7. Restriction Map of Cosmid Cos 29.3-Ali from the Alicia Rabbit

29.3-A,,

VH5

VH4

I

#VH3

I

I

” ?roba

Xbe

I

,

I

sac II

I

I

EamH I I

I

Sal

I

xpn

I

spe

I

Sfi

I

I 0

I

I

III

I

I I

,,,I

I

I

II/

I

I

I

Hind III 1 EcoR

I

II

/

I

(I!(

I

I

I

I II

I

,,I

I

,

!

I,,

/ I

/

I

I

I

, I I

/ I

I 20

I

I

I

I 10

A

I

30

40

Clone

The regions that hybridized with the VH probe are indicated by solid boxes; the region that hybridized with probe A is indicated. The sizes below the restriction map are in kb.

Cdl 968

“HI

we

we

“M

vn.

““3

-_ II II I --

“H2

_-

Pr I

“HI

1; dl” r

B I

23 kb

o&ted

]-

-13

1 5

; I”

; y’

_

_

9 cm 20.3-*I,

{

Deleted

; r”

; r” co* 18.1-M

]-

Preferential Utilization of WI The allelic inheritance of the al, a2, and a3 allotypes appears to be explained by preferential utilization of allelic VHl genes VHl-al, VHl-a2, and VHl-a3. Since these VHl genes encode prototypic al, a2, and a3 VH regions, respectively, and are preferentially utilized in VDJ rearrangements, the VHa allotype of rabbits will, for the most part, be determined by inheritance of VHl. Data from the mutant a/i rabbit strongly support the idea that a rabbit’s VH phenotype (allotype) is determined by VHl. Until at least 8 months of age, the a/i/a/i rabbit has dramatically reduced levels of a2 immunoglobulin molecules, although

___

___

Figure 9. Comparison VH3-a.2 Pseudogenes

_._

---

---

---

of Nucleotide

---

---

f

Fiaure 8. Comoarison Maps of Cos ‘842, 18.1-Ali

of Partial Restriction Cos 29.3-Ali, and Cos

The 10 kb region 3’of W/3 that is absent in Cos 29.3-Ali and Cos 18.1-Ali is indicated by brackets. The distance (kb) between the two Sfil sites in each clone is indicated below each restriction map. The VH genes are denoted by solid boxes.

kb

Discussion

_-_

COS0-e

_

l

4 5 p

to be nonfunctional, as the 5’ end of the gene was truncated. The region 5’of the codon for amino acid 18 did not resemble VH region sequence, and no leader exon or RNA splice sites could be identified. The absence of an a2-encoding VHl in a/i correlates with the loss of expression of a2 immunoglobulin. The nucleotide sequence of VH4-ali was found to be identical to the sequence of VH4-a2 of the normal a2 chromosome, and these genes appeared functional (Figure 3). Thus, although VH4-a2 appears to encode molecules with a2 allotopes (Figure 3) this gene does not appear to be readily utilized in a/i VDJ rearrangements.

---

; r”

kb

-13

““J-a,,

t

---

-_.

Sequences

.__

___

___

___

of

W/3-a/i

-_-

---

and

Nucleotides that correlate with VH sequences are in uppercase letters, and nucleotides that represent intronic or unrelated sequences are in lowercase letters. The conserved heptamer and nonamer sequences 3’of the VH region are underlined. Dashes represent nucleotide identity. Amino acid position numbers correspond to those of normal VH regions as in Kabat et al. (1987).

the levels of serum immunoglobulin are normal (Kelus and Weiss, 1988). Our studies show that Alicia rabbits are missing VHl by virtue of an apparent deletion of 10 kb encompassing VHl and VH2 and that their 3’-most VH gene, VH3, is a pseudogene. Although we cannot rule out the possibility that there are additional mutation8 in the VH chromosomal region of the Alicia rabbit, no gross deletions of this region were found by genomic blot analysis (Kelus and Weiss, 1986). Thus, it seems likely that the primary mutation in a/i is a 10 kb deletion encompassing VHl and VH2 and that the loss of VHl is responsible for the decreased expression of a2 molecules. Preferential usage of the 3’-most VH genes has been observed in VDJ rearrangements in murine and human 6 lineage cells, but in general this is limited to B cells taken early in ontogeny (Yancopoulos et al., 1984; Schroeder et al., 1987,1988). Our results suggest that B cells in both immature and adult rabbits preferentially utilize VHl. This idea is supported by the observation (Becker et al., 1990) that a JH hybridizing Hindlll fragment, identical in size to the VDJ containing Hindlll fragments from leukemic B cells, is found in Southern blots of genomic DNA from polyclonal B cells of adult rabbits. More importantly, the deletion of the VHl gene in the a/i/a/i rabbits results in the loss of most a2 immunoglobulin expression. In an a/i/al heterozygous rabbit, nearly all of the immunoglobulin molecules are al (Kelus and Weiss, 1986) and we suggest that the a/i/a’ rabbit uses VHl of the al chromosome in most of its VDJ rearrangements. We propose that, in general, if VHl on one chromosome cannot be utilized, compensation comes from VHl on the other chromosome. This idea is consistent with previous observations made in allotype-suppressed rabbits, where, for example, heterozygous alla3 rabbits exposed perinatally to anti-al antibody become suppressed for the expression of al immunoglobulin molecules (Mage et al., 1967). These suppressed rabbits compensate for the loss of al molecules by increased levels of a3 molecules (presumably from the a3 chromosome) rather than by increased levels of VHa-negative molecules from the al chromosome. In general, it seems that only in cases when VHl cannot be expressed, as in homozygous rabbits in which the VHa allotype is not expressed because of allotype suppression induced by antibody perinatally (Vice et al., 1970) or in the mutant rabbit Alicia, is the repertoire of other VH genes, such as those encoding VHa-negative molecules, extensively utilized. Why is VHl preferentially utilized in VDJ rearrange-

$titiic

Inheritance

of lmmunoglobulin

VH Allotypes

ments when there are other functional VH genes? The close proximity of VH7 to D and JH genes could be responsible for its frequent use in the VDJ recombination process. If this were the case, however, we would expect the Alicia rabbit to utilize VH4, Alicia’s 3’-most functional VH gene, preferentially; our results from analysis of cDNA clones from adult splenic mRNA show this is not the case (L. A. DiPietro, J. Short, A. Kelus, and K. L. Knight, unpublished data). Alternatively, VH7 may undergo, more effectively than other VH genes, the necessary combinatorial associations and somatic mutational events required for the generation of antibody diversity (Tonegawa, 1983). If the latter is correct, then B cells that utilized VH7 and underwent somatic mutational processes would be preferentially selected by antigen and expanded. Finally, it is possible that VH7 has some unique control elements that mediate its preferential utilization. Although VH7 seems to be the gene predominantly utilized in the generation of VHa immunoglobulin and as such would explain the allelic inheritance pattern of VHa allotypes, the expression of low levels of a2 molecules in adult a/i/a/i rabbits (Kelus and Weiss, 1986) indicates that VHa-encoding genes other than VH7 can be utilized by rabbit B cells. We do not know, however, the extent to which these other VH genes are utilized in B cells from normal rabbits. The nucleotide sequences of the V segment of VDJ genes from mature normal rabbits differ from sequences of VH7, especially within CDR 1 and CDR 2 (DiPietro and Knight, 1990). We do not know whether these differences result from the usage of multiple VH genes or from somatic hypermutation of the VH7 gene. Thus, it will be important to determine the extent to which VH7 is utilized in B cells of mature rabbits and to determine the role that genes other than VH7 may play in the expression of VHa alloytpes and in the generation of antibody diversity. VH Allotypes and Generation of Antibody Diversity The presence of allotypes in the VH region of immunoglobulin molecules had profound effects on early theories of antibody formation. Proponents of multiple germline gene theories could not explain how each member of the multigene family could encode the VHa allotype and yet have the allotypes inherited as if controlled by allelic genes. On the other hand, proponents of the somatic mutation theories could not explain how the VHa allotypic markers could be maintained during extensive somatic diversification, given that the VH allotypes reflected multiple amino acid differences. The preferential expression of one VH gene, VH7, explains the allelic inheritance pattern of VHa allotypes but raises the question as to how antibody diversity is generated. The basic organization of immunoglobulin VH, D, and JH genes is similar in higher vertebrates, but the means by which these segments are used in the generation of antibody diversity are quite different. For example, mouse and man have several hundred germline VH genes, and many of these are utilized in VDJ rearrangements; chicken, on the other hand, has only a few VH genes, and all but one are pseudogenes (Reynaud et al., 1989). It ap-

pears that antibody diversity in chicken immunoglobulins is generated by somatic mutation events, especially gene conversion, of one VH gene. Rabbit, like man and mouse, has many, apparently functional VH genes, but unlike man and mouse, it seems to rearrange predominantly one VH gene, at least for synthesis of VHa allotype molecules. We propose that much of the antibody diversity in rabbit is generated by extensive somatic diversification of VH7. Such somatic events could include hypermutation and gene conversion. Experimental Rabbits

Procedures

and Cosmid

Libraries

Homozygous rabbits of defined pedigree were maintained at Loyola University Chicago. Tissue from e/i/a/i rabbit 16137 was kindly provided by Dr. A. Kelus, Easel Institute for Immunology, Basel, Switzerland. The immunoglobulin heavy chain allotypes were determined by Ouchterlony analysis of serum (Dray et al., 1963a). DNA samples of the following homozygous rabbits were used for construction of the cosmid libraries: al/a’ = 216-3; es/as = 246D7; a/i/a/i = 16137 Cosmid libraries were prepared in the pTL5 vector (Steinmetz et al., 1962; Knight et al., 1965) with liver or spleen DNA that had been partially digested with MM. The libraries were probed with the previously described probe A (Becker et al., 1969). Clones that hybridized with probe A were probed with a pan-VH probe, ~161 (Gallarda et al., 1965); VHcontaining clones were isolated and restriction mapped. The VH cosmid clone Cos 9.2 from the $/a3 rabbit 16485 was described previously (Becker et al., 1969). A cosmid library was prepared from the leukemic B cell line PBL-1 (Knight et al., 1966). These cells were derived from a leukemic alla2 heterozygous rabbit (Becker et al., 1996) and were shown to have a functional VDJ rearrangement on the a2 chromosome (Spieker-Polet and Knight, unpublished data). The library was screened with a rabbit JH probe, pJ5 (Becker et al., 1969); pJ5+ clones were hybridized with the pan-VH probe ~161 to identify clones containing the VDJ rearrangement. Selected clones were isolated and restriction mapped; the VH genes were localized by Southern analysis (Southern, 1975). The rabbit Cp, Et.r, and Su probes were as previously described by Knight et al. (1965, 1966).

Nucleotide

Sequence

Analysis

Restriction fragments containing the VH genes were subcloned into M13mp16 or mp19 (Yanisch-Perron et al., 1965) and the nucleotide sequence was determined in both directions by the dideoxy chain termination method (Sanger et al., 1977).

We gratefully acknowledge the expert technical assistance of Mr. Shi Kang Zhai and Ms. Jessy Thomas. This research was supported by a grant from the Public Health Service, National Institutes of Health (AlMill). 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 16 U.S.C. Section 1734 solely to indicate this fact. Received

December

11, 1969.

References Becker, R. S., Zhai, S., Currier, S. J., and Knight, K. L. (1969). lg VH, DH, and JH germ-line gene segments linked by overlapping cosmid clones of rabbit DNA. J. Immunol. 142, 1351-1355. Becker, R. S., Suter, M., and Knight, K. L. (1996). Restricted utilization of VH and DH genes in leukemic rabbit B cells. Eur. J. Immunol., in press. Cebra, J. J., Colberg, J. E., and Dray, S. (1966). Rabbit lymphoid cells differentiated with respect to a-, y-, and p-heavy polypeptide chains and to allotypic markers Aal, Aa2. J. Exp. Med. 723, 547-557.

Cdl 970

Currier, S. J., Gallarda, J. L., and Knight, K. L. (1966). Partial molecular genetic map of the rabbit VH chromosomal region J. Immunol. 140, 1651-1659. DiPietro, L. A., and Knight, line VH genes and diversity Immunol., in press.

K. L. (1990). Restricted utilization of germof D regions in rabbit splenic lg mRNA. J.

Dray, S., Young, G. O., and Gerald, L. (1963a). lmmunochemical identification and genetics of rabbit y-globulin allotypes. J. Immunol. 91, 403-415. Dray, S., Young, G. O., and Nisonoff. A. (1963b). Distribution of allotypic specificities among rabbit r-globulin molecules genetically defined at two loci. Nature 199, 52-55. J. C. (1965). The molecular basis of antiProc. Natl. Acad. Sci. USA 54, 664-669.

Dubiski, S.. Dudziak. S., Skalba, D., and Dubiska, groups in rabbits. Immunology 2, 64-92.

A. (1959).

Serum

Gallarda, J. L.. Gleason, K. S., and Knight, K. L. (1965). Organization of rabbit immunoglobulin genes I. Structure and multiplicity of germline VH genes. J. Immunol. 735, 4222-4226. Johnstone, A. P, and Mole, L. E. (1917). Sequence studies on the heavy chain of rabbit immunoglobulin A of differing a-locus allotype. Biochem. J. 167, 255-267. Kabat, E. A., Wu, T. T., Reid-Miller, M., Perry, H. M., and Gottesman, K. S. (1967). Sequences of Proteins of immunologic Interest, Fourth Edition (Washington, DC.: U. S. Department of Health and Human Services, Public Health Service, National Institutes of Health). Kelus, A. S., and Weiss, S. (1966). of immunoglobulin variable regions USA 83, 4663-4666.

Mutation affecting the expression in the rabbit. Proc. Natl. Acad. Sci.

Kim, B. S., and Dray, S. (1972). Identification and genetic control of allotypic specificities on two variable region subgroups of rabbit immunoglobulin heavy chains. Eur. J. Immunol. 2, 509614. Kindt, T. J., and Capra, Plenum Press).

J. D. (1964). The Antibody

Enigma

(New York:

Kindt. T. J., and Todd, C. W. (1969). Heavy and light chain allotypic markerson rabbit homocytotropic antibody. J. Exp. Med. 134 659-666. Kindt, T. J., Mandy, W. J., and Todd, C. W. (1970). Association of allotypic specificities of group a with allotypic specificities All and Al2 in rabbit immunoglobulin. Biochemistry 9, 2026-2032. Knight, K. L., Gilman-Sachs, A., Fields, Ft., and Dray, S. (lQ7l). Allotypic determinants on the Fab fragment of rabbit As locus negative IgG-immunoglobulin. J. Immunol. 706, 761-767.

marker in the variable region of rabbit imBiochem. J. 757, 351-359.

Mole, L. E., Jackson, S. A., Porter, R. R.. and Wilkinson, J. M. (197l). Allotypically related sequences in the Fd fragment of rabbit immunoglobulin heavy chains. Biochem. J. 124, 301-316. Oudin, J. (1956a). Reaction rums d’animaux de meme 2490.

Dray, S., and Young, G. 0. (1956). Differences in the anligenic components of sera of individual rabbits as shown by induced isoprecipitins. J. Immunol. 81, 142-149.

Dreyer, W. J., and Bennett, body formation: a paradox.

Mole, L. E. (1975). A genetic munoglobulin heavy chain.

de precipitation specifique entre des seespece. C. R. Acad. Sci. [D] 242, 2469-

Oudin, J. (1956b). L’Allotypie de certains antigens rum. C. R. Acad. Sci. [D] 242, 2606-2606.

proteidiques

du se-

Pernis, B., Chiappino, M. B., Kelus, A. S., and Gell, P G. H. (1965). Cellular localization of immunoglobulins with different allotypic specificities in rabbit lymphoid tissues. J. Exp. Med. 122, 653-676. Reynaud, C.-A., Dahan, A., Anquez, V., and Weill, J.-C. (1969). Somatic hyperconversion diversifies the single Vu gene of the chicken with a high incidence in the D region. Cell 59, 171-163. Sanger, F., Nicklen, S., and Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463-5467 Schroeder, restriction

H. W., Jr., Hillson, J. L., and Perlmutter, R. M. (1967). Early of the human antibody repertoire. Science 238, 791-793.

Schroeder, H. W., Jr., Walter, M. A., Holker, M. H., Ebens, A., Willems Van Dijk, K., Liao, L. C., Cox, D. W., Mimer, E. C. R, and Perlmutter, R. M. (1966). Physical linkage of a human immunoglobulin heavy chain variable region gene segment to diversity and joining region elements. Proc. Natl. Acad. Sci. USA 85, 6196-9200. Southern, E. (1975). Detection of specific ments separated by gel electrophoresis.

sequences among DNA fragJ. Mol. Bid. 98, 503-517.

Steinmetz, M., Winoto, A., Minard, K., and Hood, L. (1962). Clusters of genes encoding mouse transplantation antigens. Cell 28, 469-496. Strosberg, A. D. (1977). Multiple of the iceberg? Immunogenetics

expression of rabbit 4, 449-513.

Todd, C. W. (1963). Allotypy in rabbit Res. Commun. 17, 170-175. Tonegawa. S. (1963). 302, 575-561.

Somatic

19s protein.

generation

of antibody

allotypes:

Biochem. diversity.

the tip Biophys. Nature

Vice, J. L., Gilman-Sachs, A., Hunt, W. L., and Dray, S. (1970). Allotype suppression in ezaz homozygous rabbits fostered in uteri of a2immunized alal homozygous mothers and injected at birth with antia2 antiserum. J. Immunol. 104, 550-554. Yancopoulos, G. D., Desiderio, S. V., Paskind, M., Kearney, J. F., Baltimore, D., and Alt, F (1964). Preferential utilization of the most JH proximal Vu gene segments in pre-B cell lines. Nature 377, 727-733. Yanisch-Perron, C., Vieira, J., and Messing, J. (1965). Improved Ml3 phage cloning vectors and host strains: nucleotide sequences of the M13mp16 and pUC19 vectors. Gene 33, 103-119.

Knight, K. L., Burnett, R. C., and McNicholas, J. M. (1965). Organizalion and polymorphism of rabbit immunoglobulin heavy chain genes. J. Immunol. 134, X245-1250.

GenBank

Knight, K. L., Spieker-Polet, H., Kazdin, D. S., and Oi, VT. (1966). Transgenic rabbits with lymphocytic leukemia induced by the c-myc oncogene fused with the immunoglobulin heavy chain enhancer. Proc. Natl. Acad. Sci. USA 85, 3130-3134.

The accession numbers for the sequences published in this paper are JO4664JO4670. The accession numbers for the sequences referred to, but not published, in this paper are JO467l-JO4679.

Landucci-Tosi, S., Mage, R. G., and Dubiski, S. (1970). Distribution allotypic specificities Al, A2, A14, and Al5 among immunoglobulin molecules. J. Immunol. 104, 641-647.

Note

Lennox, E. S., and Cohn, them. 36, 365-406.

M. (1967). Immunoglobulins.

Annu.

of G

Rev. Bio-

Mage, R. (1981). The phenotypic expression of rabbit immunoglobulins: a model of complex regulated gene expression and cellular differentiation. Contemp. Top. Mol. Immunol. 8, 69-112. Mage, R. G., Young, G. O., and Dray, S. (1967). An effect upon the regulation of gene expression: alfotype suppression at the a locus in heterozygous Offspring of immunized rabbits, J. Immunol. 98, 502509. Mage, R. G., Bernstein, K. E., McCartney-Francis, N., Alexander, C. B., Young-Cooper, G. O., Padlan, E. A., and Cohen, G. H. (1964). The structural and genetic basis for expression of normal and latent Vua allotypes of the rabbit. Mol. Immunol. 27, 1067-1061.

Accesslon

Added

Numben,

in Proof

The work referred to in this paper as DiPietro et al., unpublished data, is now in press. DiPietro, L. A., Short, J. A., Zhai, S., Meier, D., Kelus, A., and Knight, K. L. (1990). Limited expression of immunoglobulin VH in the mutant rabbit “Alicia:’ Eur. J. Immunol., in press.