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ImmunogeneHcs 35: 131-135, 1992

genetics

© Springer-Verlag 1992

A new

VH-CI recombinant

in the rabbit

Rose G. Mage 1, Glendowlyn O. Young-Cooper 1, Cornelius B. Alexander 1, W. Carey Hanly 2, and Barbara A. Newman ~ t Laboratoryof Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA 2 Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago, IL 60612, USA Received June 17, 1991

In the rabbit, there are genetically controlled differences in immunoglobulin heavy chain constant (C) regions (C H allotypes), as well as variable (I0 region genetic markers (e. g., Vna allotype-encoding genes; reviewed in Mage 1986). The Vna allotypes behave as if controlled by allelic genes; in normal rabbits, the majority of immunoglobulins bear detectable VHa allotypes (reviewed in Mage et al. 1984). These two facts may be explained by the recent observations that the most J-proximal Vn gene, (called Vnl), encodes a typical Vna allotype protein sequence (Knight and Becker 1990) and that the VH1 gene appears to be preferentially rearranged in a large proportion of rabbit B cells (Becker et al. 1990; Allegrucci et al. 1991). It was the availability of the serologically defined allotypes that allowed demonstration of the linked inheritance of VH and CH genes, a n d contributed to the definition of Igh haplotypes, i. e., the clusters of linked VH and Ca genes that constitute the complex Igh locus (Mage et al. 1982). Although haplotypes are generally inherited intact, recombinations between rabbit Vn and Cn markers have been observed. The first such observation was reported in 1971 (Mage et al. 1971); since that time, eleven other recombinants have been reported (Kindt and Mandy 1972; Hamers-Casterman and Hamers 1975; Kelus and Steinberg 1991). Here, we report that we have observed a thirteenth recombination and have developed a strain o f rabbits with the new recombinant haplotype,

R2M. Although the allotypes were originally defined serologically, more recently, DNA probes have been developed which allowed detection of restriction fragment length polymorphisms (RFLPs) that correlate well with serologically defined haplotypes and have helped us to localize the sites of recombination in three recombinants, Address correspondence and offprint requests to: R.G. Mage.

(RIM, R3K, and R4K; Newman et al. 1991). In all three, the sites map 3' of the most JH-proximal VH gene (VH1) and 5' ofJ H (Newman et al. 1991). Thus far, two recombinants, R3K (F-C) and R4K (H-I), map to sites toward the 5' end of the D H region and one, RIM (F-I), toward the 3' end. Another recently described recombinant, R7K [H-(FI)] (Kelus and Steinberg 1991), clearly differs from the first three characterized at the DNA level. In the R7K, both serological and DNA mapping studies indicate that the site of recombination is 3' of the C~t gene; further characterization of the site of the R7K recombination is currently in progress (B. A. Newman, C. B. Alexander, D. Meier, A. S. Kelus, W. C. Hanly, and R. G. Mage). The most recent recombinant, reported here, is R2M which carries a chromosome resulting from recombination between a sire' s R3K (F-C) and I haplotypes, yielding the new haplotype [I-(F-C)]. As shown in Figure 1, the R3K haplotype was itself derived from a recombination which occurred in the colony of the Basel Institute for Immunology; specifically, the new haplotype, R3K or F-C (V~a2, msl7, d l l , el5, f72, g74), resulted from a recombination between haplotypes F(V~a2, msl 7, d12, e15, f69, g77) and C(Vnal, msl7, d l l , e15, f72, g74) in the male parent of the proband rabbit (Kelus and Steinberg 1991). The proband doe was bred to a homozygous male ofB haplotype. Brother-sister matings between two of the progeny produced the heterozygous male breeder 1 i583 (haplotypes B/R3K) which was sent to the National Institutes of Health (NIH) by A.S. Kelus along with a homozygous R3K/R3K sibling (11901). Table 1A and B summarize further the results of matings that were conducted in order to maintain the R3K haplotype at the NIH and that were informative for Igh locus recombination. Rabbit 11583 was initially outbred to five different does generating 22 weaned progeny, ten of which inherited the R3K chromosome. Fourteen additional weaned progeny

R.G. Mage et al. : A new Vn-CHrecombinant in the rabbit

132 VHa Cpms C~'de CcLfg H a3-16-11-15-72-74

Fa2-17-12-15-69-77

B a1-17-12-15-71-75~ - B a1-17-12-15-71-75

F a2-17-12-15-69-77

N3K 11583I BZ251-3 I a1-17-12-14-69-77~ ]" a1-17-12-14-69-77~,.,,"

+ 2AA303-2

3AA274-2 i~ 7 (F-C)R3KY//a~1~i~7 1AA91-6+ BB258

I

(F-C)R3K ~//_-~7_i:~i:i~]5~72~ R2M Fig. 1. Pedigree showing the derivation of the R2M recombinant bred at the NIH, from the R3Krecombinant obtained from the Basel Institute for Immunology.Key haplotypes are shown with Vna, Cg ms, C3~de, and Caf and g allotypes indicated. Dashes signify the region within which serologicaldata do not permit distinction betweenthe two parental haplotypes. Only progeny relevant to the R2Mlineage are shown. Additional R3K rabbits were obtained from homozygous male 11901 and from matings of heterozygousmale 11583 to six different females. The results of R3Kand R2M breeding are summarizedin Table 1 (A, B) and Table 1 (C, D), respectively.

were obtained in two litters from matings of this male with one of his offspring (2AA303-2) among which was the homozygousR3Kfemale 1AA91-6 (Fig. 1). In all of these initial backcross and F 2 progeny, the R3K chromosome was inherited intact; that is, the VHa2 and C3,dll allotypes were inherited together (a total of 23 informative gametes). In the next generation, however, proband BB258-2 (Fig. 1), one of four offspring of the mating of the homozygous R3K female 1AA91-6 with the heterozygous male 3AA274-2 (R3K/1), appeared to have inherited neither of the complete haplotypes from the sire [I(Vnal, msl7, d12, e14, f69, g77)/R3K (Vna2, ms17, d11, e15, y'72, g74)]. In the serum of the V~a heterozygote we found al and a2 allotypes, d l l , e15, f72, g74 but no d12, e14, f69 or g77, all of which should have been inherited

with the VHal. Indeed, heterozygous littermate BB258-1 inherited the complete I haplotype; littermates BB258-3 and -4 inherited the complete R3K haplotype from the sire (Fig. 1). Thus proband BB258-2 appeared to have a chromosome resulting from a recombination between the sire's I and R3K haplotypes with the Vn, or 5' portion of the new haplotype from I and the C3, region from R3K. The occurrence of a new recombination, denoted R2M, [I-(F-C)], so soon after the R2K recombination event, raised the possibility that there is unusual instability in the R3K chromosome. In this regard, we have conducted additional breedings to maintain both the R3K and the R2M recombinant chromosomes; no further recombinants have been observed thus far (Table 1). A total of 167 weaned progeny in 34 backcross and F2 litters were informative for Vn-CH recombination between the R3K haplotype and another haplotype at the Igh locus (Table 1A and B); for the R2M haplotype, a total of 93 weaned progeny in 21 backcross and F 2 litters were informative for Vn-CH recombination (Table 1C and D). The breeding of R2M to homozygosity was particularly difficult because of apparent deficiency in the numbers of weaned females carrying the R2M recombinant chromosome. During the initial outbreeding to develop this strain, only four of the first 62 weaned backcross progeny were R2M females and one of these died prior to bearing any progeny (Table 1C). In this group, the R2M chromosome was found in 54.3 % of the weaned males and in only 14.8 % of the weaned females, whereas in the comparable group from R3Kbreeding, the R3K chromosome was found in 55.2% of the males and 44.6% of the females. It is not known whether this deficiency in R2M females represented transmission distortion or selective loss of females carrying the R2M chromosome prior to weaning. We compared mean litter sizes at birth and at weaning in the 15 backcross litters of the R2M group with 18 backcross litters in the R3K group that were bred and weaned in the same environment during the same period of time. Both at birth and at weaning, the mean litter sizes were not significantly different in the R2M and R3K groups (R2M 6 . 6 0 + / - . 9 0 , R3K 6 . 7 8 + / - . 7 1 born; R2M 4 . 1 3 + / - . 7 3 , R3K 4 . 8 3 + / .68 weaned). Following successful breeding of R2M homozygotes, we analyzed genomic R2M DNA to localize the site of the recombination more precisely than is possible by serological tests. The methods and the probes used for these studies have been described recently (Newman et al. 1991). The results of the Southern analyses for RFLPs used to determine which portions of the parental I and R3K chromosomes were found in the R2M recombinant are shown in Figure 2A. The sources of the probes used are shown diagrammatically beneath the map in Figure 2B. Restriction fragment lengths characteristic of the D N A of the 5' haplotype I were found in Hin dlII digests hybridiz-

R.G. Mage et al.: A new Vn-CH recombinant in the rabbit

133

Table 1. Summary of breeding of the R3K and R2M Vff-CH recombinant haplotypes at the NIH. Number of litters

R3K A

Backcrosses* 25

B

F2*

R2M C D

9

Total progeny born

Total progeny weaned

R3/R3

163

114

64

53

0 (0)* 11 (13.25)

57 (57) 27 (26.5)

56 (57) 15 (13.25)

R2/R2 0 (0) 10 (7.75)

R2/23 (31) 15 (15.5)

- /39 (31) 6 (7.75)

Backcrossesu 15

99

62

F2t

46

31

6

R3/-

- /-

%c. recombinants

1 0

* Heterozygotesx homozygotes; in 23 litters the male was the heterozygous parent and in two the female was heterozygous. t Heterozygotesx heterozygotes. * Numbers in parenthesesindicate expected values for Mendelian segregation. In the R2M backcross group only, there is a significant deviation from expected Mendelian segregation of the alleles (p< .05, X2 test). II Heterozygotesxhomozygotes; in 14 litters the male was heterozygous and in one the female was heterozygous.

ed with a probe (A) from a region 5' of known DH genes and - 7 . 5 kilobase (kb) 3' of VH1 (the most J-proximal VH gene; Figure 2A panel A). The D N A of R2M also resembles the 5' parental I haplotype when Hin dIII (Fig. 2A, panel B), Msp I or Taq I digests (not shown) were hybridized to the DH2 probe that identifies R F L P in the DH2 regions. The same was true when the PD4-H-B probe, which detects RFLP in the region 3' of D2b was hybridized to Hin dlII digests (Fig. 2A, panel C) or Pvu II digests (data not shown). As shown in panel D, the DH1 probe detects a Did-containing Hin dIII fragment of 10 kilobase (kb) characteristic of haplotype I and not the 17 kb fragment characteristic of haplotypes C and F-C (R3K). This result extends the similarity of R2M to its 5' parent I at least to a Hin dlII site 3' of the Dld gene. R2M resembles its 3' parent, R3K, in the JH and Cg regions as shown in Figure 2A panels E and F, where R F L P in digests with Bgl II and Hin dlII were identified with JH and Cg probes, respectively. These results localize the R2M site of recombination 3' of the D2b and Dld genes and - 5 kb 5' of Jn where the 5' Bgl II site is located, and resemble those obtained for the recombinant, RIM (F-I; Newman et al. 1991). We were, however, able to narrow down the region within which the R2M recombination occurred more than was possible in the studies of the first three recombinants. The site of recombination in R2M is clearly different from that localized for the parental R3K haplotype (and the unrelated R4K haplotype) which map 5' of D2a (Fig. 2C). Rather, the R2M recombination site is similar to that of RIM and localizes in or near regions containing highly repetitive stretches of D N A (Newman et al. 1991). The

R3K and R4K sites of recombination also are localized to regions containing repetitive D N A (Newman et al. 1991). It is of interest that the distal end of the long arm of human chromosome 14 (14q32) has a polymorphic VNTR locus (IGHC) 5' of the JH region (Silva et al. 1987). The frequency of genetic recombination in this region is high ( > 60 cM) compared to its physical size (10 cM predicted; Nakamura et al. 1989). Some translocations in human lymphoid malignancies have been reported to involve sites in this vicinity (Bakhshi et al. 1985; Haluska et al., 1987). Such translocations may be mediated by the recombinase enzymes involved in site specific Igh and T-cell receptor gene rearrangements; the repetitive D N A sequences may contribute by providing open chromatin structure needed for accessibility of recombinases (Boehm et al. 1989). All of the V~CH recombinations reported in rabbits, including the present R2M recombination, occurred in the male parent. Kelus and Steinberg (1991), have suggested that these recombinations occurred in mitosis during spermatogenesis. Interestingly, there is a significantly higher recombination frequency in males for the intervals involving the human Igh locus (IGHC) and other markers in the human 14q32 region. Whether Igh-specific recombinase activity is present during any stages of spermatogenesis remains to be determined. In summary, we report here the thirteenth laboratory observation of a V~CH recombination (R2M) in the rabbit. All thirteen recombinations have occurred in the male parent. The R2M recombination site maps 3' of the VH genes and 5' of the JH genes as did the sites of three previously characterized recombinations (Newman et al. 1991). The R2M site maps 3' of the Dld DH gene which

R.G. Mage et al.: A new VtFCH recombinant in the rabbit

134

A.

B. r•jl

/

10

20

30

40

50

60

i

i

i

i

i

i

(VH)n=o'VH1

--//"I

Ola

II HX H A

Dlb

D2aDlcD2b

II Ill Ill I I II I H SaE SS M H DH1 DI_I2

C. [I-(F-C)I Recombinant R2M

I

F

.

Dld

F-I Recombinant R1M

HB M PD4 H-B

70 i

JH

C# m

I Illll I11" H BBBE HHB I I III S'J H BeE JH E

R3K F-C

I

Fig. 2A-C. A Results of Southern analyses of restriction enzyme digests of DNA from the recombinant R2M and from parental R3K and I haplotypes, hybridized with a series of DNA probes which permit localization of the site of the recombination. The sources of the probes, which have been described previously (Newman et al. 1991) are indicated below the map in Fig. 2B. The Jn probe used in panel E was a - 2 kb Barn HI-Barn HI fragment. B Diagrammatic representation of the Igh genetic region showing the sources of the probes used in Fig. 2A. Enzyme abbreviations are: H, Hin dlII; X, Xba I; B, Barn HI; Sa, Sal I; S, Sac I; E, Eco RI. C Diagrammatic summary of the region within which the R2M site of recombination is localized with reference to the map above. The sites determined earlier for three other recombinants (Newman et al. 1991) are shown for comparison.

is t o w a r d the 3' e n d o f t h e D m c o n t a i n i n g r e g i o n a n d is p r o b a b l y in o r n e a r s t r e t c h e s o f r e p e t i t i v e D N A . A s p r e v i o u s l y s u g g e s t e d ( N e w m a n et al. 1991), t h e r e m a y b e t w o or m o r e " h o t - s p o t s o f r e c o m b i n a t i o n " w i t h i n or

n e a r stretches o f r e p e t i t i v e D N A in t h e D H - c o n t a i n i n g r e g i o n o f t h e rabbit. A l t h o u g h t h e R 2 M r e c o m b i n a t i o n occ u r r e d i n the c o u r s e o f b r e e d i n g a n e a r l i e r r e c o m b i n a n t c h r o m o s o m e , R 3 K , t h e r e c o m b i n a t i o n sites in t h e s e t w o

R.G. Mage et al.: A new Vh,-C~ recombinant in the rabbit r e c o m b i n a n t s m a p o n o p p o s i t e side o f t h e D n - c o n t a i n i n g r e g i o n b u t b o t h a r e in o r n e a r r e g i o n s o f r e p e t i t i v e D N A . Acknowledgments. We thank Dr. Andrew S. Kelus, Basel Institute for Immunology, for the gift of R3K recombinant rabbits, Dr. Katherine L. Knight and Dr. Robert S. Becker, Loyola University of Chicago, for gifts of subclones and cosmids from which many of our probes were derived, Dr. David Alling, NIAID, NIH for assistance with statistical analyses, Marlon Hall for help in serum typing and Ms Shirley Starnes for editorial assistance.

References Allegrucci, M., Young-Cooper, G.O., Alexander, C.B., Newman, B. A., and Mage, R. G. : Preferential rearrangement in normal rabbits of the 3' Vi~a allotype gene that is deleted in Alicia mutants; somatic hypermutation/conversion may play a major role in generating the heterogeneity of rabbit heavy chain variable region genes. Eur J Immunol 21: 411-4-17, 1991 Bakhshi, A., Jensen, J. P., Goldman, P., Wright, J. J., McBride, O. W., Epstein, A.L., and Korsmeyer, S.J.: Cloning the chromosomal breakpoint of t (14; 18) human lymphomas: clustering around JH on chromosome 14 and near a transcriptional unit on 18. Cell 41: 899-906, 1985 Becker, R. S., Suter, M., and Knight, K. L.: Restricted utilization of VH and D H genes in leukemic rabbit B cells. Eur J Immunol 20: 397-402, 1990 Boehm, T., Mengle-Gaw, L., Kees, U.R., Spurr, N., Lavenir, I., Forster, A., and Rabbits, T. H. : Alternating purine-pyrimidine tracts may promote chromosomal translocations seen in a variety of human lymphoid tumors. EMBO J 8: 2621-2631, 1989 Halnska, F.G., Tsujimoto, Y., and Croce, C.M.: Mechanisms of chromosome translocation in B- and T-cell neoplasia. Trends Genet 3: 11-15, 1987 Hamers-Casterrnan, C. and Hamers, R.: A second crossing-over between d and a locus in rabbit immunoglobulin 3' chain. Immunogenetics 2: 597-603, 1975

135 Kelus, A. S. and Steinberg, C. M.: Is there a high rate of mitotic recombination between the loci encoding immunoglobulin VH and CI¢ regions in gonial cells? lmmunogenetics 33: 255-259, 1991 Kin&, T. J. and Mandy, W. J.: Recombination of genes coding for constant and variable regions of immunoglobulin heavy chains. J Immunol 108: 1110-1113, 1972 Knight, K.L. and Becker, R.S.: Molecular basis of the allelic inheritance of rabbit immunoglobulin Vr~ allotypes: implications for the generation of antibody diversity. Cell 60: 963-970, 1990 Mage, R. G. : Rabbit immunoglobulin allotypes. In D. M. Weir, L. A. Herzenberg, C. C. Blackwell, and L. A. Herzenberg (eds.): Handbook of Experimental Immunology 3, 4th Edition, pp. 99.1-99.25, Blackwell Scientific, Oxford, 1986 Mage, R.G., Young-Cooper, G.O., and Alexander, C.B.: Genetic control of variable and constant regions of immunoglobulin heavy chain. Nature, New Biology 230: 63-64, 1971 Mage, R.G., Dray, S., Gilman-Sachs, A., Hamers-Casterman, C., Hamers, R., Hanly, W. C., K.indt, T.J., Knight, K.L., Mandy, W.J., and Naessens, J. : Rabbit heavy chain haplotypes-allotypic determinants expressed by Vn-CI4recombinants. Immunogenetics 15: 287-297, 1982 Mage, R.G., Bernstein, K.E., McCartney-Francis, N., Alexander, C.B., Young-Cooper, G.O., Padlan, E.A., and Cohen, G.H.: The structural and genetic basis for expression of normal and latent VHa allotypes of the rabbit. Mol lmmunol 21: 1067-1081, 1984 Nakamura, Y., Lathrop, M., O'Connell, P., Leppert, M., Kamboh, M.I., Lalouel, J.M., and White, R.: Frequent recombination is observed in the distal end of the long arm of chromosome 14. Genomies 4: 76-81, 1989 Newman, B.A., Young-Cooper, G.O., Alexander, C.B., Becker, R. S., Knight, K.L., Kelus, A. S., Meier, D., and Mage, R. G.: Molecular analysis of recombination sites within the immunoglobulin heavy chain locus of the rabbit. Immunogeneties 34: 101-109, 1991 Silva, A.J., Johnson, J.P., and White, R.L.: Characterization of a highly polymorphic region 5' to Ja in the human immunoglobulin heavy chain. Nucleic Acids Res 15: 3845-3857, 1987