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Genetic polymorphism of rabbit VHa region: A new allotype, a108
~) INSTITUT PASTEUR/ELSEVIER Paris 1989
Res. l"m. m. .u .n .o .l .. . 1989, 140, 265-273
GENETIC POLYMORPHISM OF RABBIT Vna REGION: A NEW ALI.DTYPE, a108
H. Haouas (l) (,), A. E! Gaaied (2) and P.-A. Cazenave (l) (1) Unitd d'lmmunochimie Analytique, Ddpartement d'Immunologie, Institut Pasteur and LA CNRS 359 75724 Paris Cedex 15,
and (2) Laboratoire d'Immunogdndtique, Ddpartement des Sciences Biologiques, Facultd des Sciences de Tunis, Tunis
SUMMARY We report a new rabbit IgV H allotype, designated a108, which was detected following studies of the progeny of a wild rabbit (Oryctolagus cuniculus) from Zembra Island (Tunisia). This allotype seems to be specific of this island, since it has not been detected in other wild rabbit populations (in Spain, Portugal or France). Its determinants are located on the Fd fragment of the immunoglobulin (Ig) heavy chain and it behaves like the product of an allele at the a locus. Allotype a108 was strongly related serologically to al and a107, and to a lesser extent to al01, a109 and a3 allotypes. We also detected determinants shared among the al, a107, a108, al01 and a109 allotypes. These determinants were expressed by a large fraction of Ig molecules from rabbits of allotype al, a107 and a108 and by a very small fraction of Ig from rabbits of allotype al01 and a109. KEY-WORDS" Allotypy, VHa region, Immunoglobulin; New allotype a108, Rabbit. INTRODUCTION VH allotypes of rabbit immunoglobulins (Ig) reflect genetically controlled alternative structures of the Vri-gene segment 0VIage, 1986; Cazenave, 1981). Consequently, they are a valuable tool for understanding the evolution of VH genes in this species. Defined by specific serological reagents, the VH
Submitted April 3, 1989, accepted April 7, 1989. (*) To whomcorrespondence should be addressed.
266
H. H A O U A S E T A L .
allotypes have been subdivided into four subgroups, VHa, VHX, VHY and VHW, which are encoded by four VH-gene families: a, x, y, w (Mage, 1986; Knight et al., 1971 ; Roux, 1981, Roux et al., 1981). The VHa subgroup represents the majority of Ig, and the VHa-negative subgroups (VHx, VHY, VHW) only 10 to 30 070 of the total Ig. In domestic rabbits, three allelic forms of VHa subgroup: al, a2, a3, have been identified (Oudin, 1960a). Each of these allotypes appears to be a family of variants (Oudin, 196{)5) which differ in a few amino acid substitutions (Margolies et al., 1977). They appear to be encoded by closely linked genes (Oudin, 1960b) and are inherited together in the same proportion in sera of non-immunized rabbits (Oudin, 1960b; Roland et al, 1977). Studies of Ig heavy chains in wild rabbits, however, reveal that the polymorphism observed in the domestic rabbit represents only a fraction of the genetic poiymorphism of O. c~aniculus species. TMs is apparent because additional allotypes al00 to a109 have been detected in wild rabbit pop~ations in France, Spain, Portugal and Tunisia (Cazenave et al, 1974; Brezin and Cazenave, 1980; Brezin et al., 1979; Haouas et aL, 1987). In this paper, we describe the a108 ailotype found in the population of Zembra Island (Tunisia) and characterize its serological relatedness with different allotypes of the " a " series. In addition, we show that this allotype comprises a family of variants.
MATERIALS AND METHODS Purification of IgG, Fab and heavy and light chains.
IgG were purified from non-immune sera on DEAE cellulose (Levy and Sober, 1960). Fab was purified from IgG by papain hydrolysis (Porter, 1959). Heavy and light chains were separated by gel filtration after reduction and alkylation of IgG (Fleishman et al, 1962). Antisera and antibodies.
Anti-allotype antisera were prepared as previously described (Br~zin and Cazenave, 1975). Antibodies were isolated from antiallotypic antiserum on immunosorbent columns of IgG (Cambiaso et al., 1975). The antibodies adsorbed by passage through the Sepharose AH-4B immunosorbent column, were eluted with 0.2M glycine-HCl buffer pH 2.2 containing 0.SM NaCI. The eluate was immediately neutralized using 1M Tris buffer pH 9.
Ig = immunoglobulin.
[ RIA = radioimmunoassay.
GENETIC
POL YMORPHISM
OF RABBIT
Vfta R E G I O N
267
Fracfionation of IgG. Sepharose AH-4B coupled to antibody H620 (anti-al08 allotype serum) was incubated 2 h with al01 or a109 IgG (Cambiaso et al., 1975). The unboud fraction was removed by several washings with Tris (0.2M)/NaCI (0.SM) pH 8. The bound fraction was eluted by HCl/glycine/NaCl buffer. The same experiment was repeated until no protein was detected in the eluted fraction.
Radioimmunoassays (RIA). Reactions of the allotype with anti-allotype antibodies were studied by two RIA assays: liquid-phase RIA using insolubilized antisera (Landucci-Tosi and MaRe, 1970) and a solid-phase RIA using polyvinyl microtitre plates (Pierle aad Kliman, 1976). Antisera were insolubilized by polymerization with ethylchloroformate (Avrameas and Ternynck, 1967). IgG and Fab were 125I-labelled according to the Greenwood method (Greenwood et al., 1963).
Animals. The wild rabbits used were from Zembra Island (Tunisia). Domestic rabbits were Bouscat Giant.
RESULTS
Description of the a108 allotype. A m o n g the wild rabbits trapped on the Island of Zembra, one of them (LG896) carrying the VHa2 allotypic specificity was mated with a domestic doe homozygous a 3 / a 3 (fig. 1). None of the three offspring inherited the a2 aUotype f r o m the father; each exhibited the a3 +, a 2 - phenotype. One of
IN
L6896
I
®
I .-'H461 e~ i H747 FIo. I. - - Pedigree of LG 801 rabbit family.
C) = a2; A = a3; • = a108.
268
H. H A O U A S E T AL.
the litter (H461) was crossed with an a2/a3 domestic male. All offspring received the a3 allele from the buck. One of the males (H626) was randomly chosen and backcrossed with the H461 female. In seven progeny obtained, two produced sera that precipitated neither with anti-a3 sera nor with any antisera directed against the other allotypic specificities of the " a " series (al, a2, al00, al01, a106, a107, a109). This particular behaviour suggested that these individuals could be homozygous for an unknown allele at the a locus. Hence, an immune serum was prepared in an a3/a3 domestic rabbit against the IgG isolated from the serum of one of these individuals (H747). The av~iserum obtained was able to precipitate the immunizing IgG but was non-reactive with all known allotypes of the a series. Sera of all the family members were studied for their reactions with this antiserum by precipitation in liquid medium and RIA. Analyses si~owed that it behaved as an antiserum directed ag~nst an unknown aUotype of the a series, and we designated it a108 (fig. 1). The results summarized in figure 2 demonstrate that the a108 allotype is located on the heavy chain and Fab fragment.
.o//,o/
600
imm
in -m .elm .,C C: m
• IgG oH
50-
<>H reass
40-
• Fab AL
302010l
;
4.10-4
|
_/ i
2
4.t0"
;
A
l
4 nMIml
F I G . 2. - - Inhibition o f the binding o f 1251-labelled a108 + lgG to anti-al08 serum by IgG, Fab, heavy and light chains purified from H747 serum, and heavy chains reassociated with light chains isolated from b 4 / b 9 3 rabbit serum.
GENETIC POL YMORPHISM
OF RABBIT
Vh.a R E G I O N
~N
2v;,
TABLE I. - - Binding o f radiolabelled F a b to different antisera. Fab
Antisera
al
a2
a3
Anti-al (al01/al01) Anti-a2 (a3/a3) Anti-a3 (a2/a2) Anti-al00 (al01/al01) Anti-al01 (al/a3) Anti-al06 (al/a3) Anti-al07 (a2/a3) Anti-al08 (a3/a3) Anti-al09 (a3/a3)
82 . 75 58 .
70 -
62 . 98 47 . -
-
.
. . . .
. . .
allotype
(070)
al00 al01 a106 a107 a108 a109 71
-
.
. 58 91
. . .
76 . 55 89 . 84
. 76 -
82 . .
76 . 32 .
. . .
.
.
. 79 63
.
80
48
44 . 34 80
69 -
.
93
= binding below 25%.
a108 cross-reactivities.
The cross-reactivities of the various a allotypes were examined by binding 125I-labelled Fab from each of the allotypes to antiaUotypic antibodies against these various allotypes. As shown in table I, anti-al08 reacted with al and a107 allotypes, and conversely, anti-al, anti-al07 and anti-a3 reacted with a108 Fab. It is noteworthy that only a portion of the molecules (variants) of these allotypes were involved in several of these cross-reactivities. These results indicate that a108 molecules are comprised of two l=,opulations of
o am7 eat08 = al
//
/.
,, a!09 • aiOI oa2 • alO0 oa3
0
A
•
Dilution of inhibitor F [ o . 3. - -
Inhibition by different allotypic sera of the binding of radioiabeilcd a108 + Fab tt; purified homologous anti-allotypic antibodies.
270
H. H A O U A S E T AL.
molecules, one which reacts with anti-al07 (and/or anti-a3) and one which does not react with anti-al07 (or anti-a3). The a108 cross-reactivities have been studied in greater detail, using inhibition of the binding of al08+-labelled Fat) to homologous immune serum by different allotypes. As shown in figure 3, the reaction was inhibited by al, a107, al01 and a109 allotypes. These results show that the al and a107 allotypes carry most of the allot,~q~ic determinants recognized by the anti-al08 antiserum. However, the al and a107 allotypes were not precipitated by the anti-al08 antibodies. We concluded that these determinants have a different molecular distribution in the al, a107 and a108 allotypic families of variants. The results of additional experiments are in agreement with this interpretation. For instance, the a108 al!otype totally inhibited the binding of al+-and al07+-labelled Fab to homologous sera (data not shown). In another step, a108 cross-reactivities have also been studied by isolation of anti-al and anti-al07 antibodies capable of binding to al and a108 allotypes respectively. Inhibition of the binding of al08+-labeUed Fab to these isolated cross-reacting heterologous antiallotypic antibodies was studied and confirmed previous results, and, in addition, revealed that at least some of the allotypic determinants involved in al, a107 and a108 cross-reactivities are also present in a3, MOO, a106, al01 and a109 allotypic families.
. o ..-.
oa101la108+1 "~_
" :] i~/A~l/t
• all~
Aa3 .. ®al ,,.a. [ai08-I
.
Inhibitor (pg/ml) FIG. 4. - - Inhibition by different al!otypic sera o f the binding o f radiolabelled al + (A) or a107 + (B) Fab to anti-al08 antibodies purified on homologous allotype.
GENETIC POL YMORPHISM OF R A B B I T Vna REGION
271
TABLE II. - - I n h i b i t i o n by d i f f e r e n t allotypes of the binding of labelled F a b to homologous a n t i a l l o t y p i e sera. A l l o t y p e inlfibitor ( % ) Antisera
al
a2
a3
Anti-al08 (a3/a3) Anti-a1 (al01/al01) Anti-al07 (a3/a3)
99 89 84
- (*) -(**) -(**)
. .
al00
al01
a106
a107
a108
a109
-
96 . .
-
98 68 82
99 87 85
89 -
. .
. .
(*) inhibition below 20070 with a 10 -2 dilution of inhibitor serum. (**) Inhibition below 20070 with a 10-i dilution of inhibitor serum.
It was shown that the a101 and a109 allotypes did not bind to anti-al08 serum (table I) but were able to inhibit the binding of a108 allotype to homologous antiallotypic antibodies (table II) suggesting that only a small fraction of Ig molecules bearing the al01 and a109 determinants is responsible for the cross-reactivity with a108. As shown in figure 4, the a108 determinants involved in these cross-reactivities are those involved in the cross-leactivities with al and a107 allotypes.
DISCUSSION
The evidence presented in this paper indicates that the a108 allotype belongs to the a series. This allotype found on Zembra Island (Tunisia) has not been detected in other wild rabbit populations frcm France, Spain and Portugal (data not shown). However, the a108 aUotype has extensive cross-reactivity with allotypes having different geographical distributions: al and a3 (present in domestic rabbits and in several wild populations), al00 (present in France, Spain and Zembra), al01 (detected in France but not in other countries), a106 (particular to Portugal), a107 (present in Spain, Portugal and Zembra) and a109 (present in Spain and Portugal) (Haouas et al., 1987). These cross-reactivities reflect the complex evolution of the a allotypes. Understanding of the mechanisms of this evolution is clouded by the number of genes analysed which belong to the VH multigene family (Gallarda et al., 1985) and by the nature of the wild populations studied for which a "founders effect" is probably crucial, if one considers the natural history of the O. cuniculus species (Lopez-Martinez, 1977). It was shown that a major variant (or subfamily of variants) is present at a similar high concentration in the sera of al +, a107 + and a108 + rabbits. The same variant (or subfamily of variants) is detected, at a very low level
H. H A O U A S E T AL.
272
(1% of the allotype) in al01 + and a109 + sera. This observation suggests that either the n u m b e r o f VH genes corresponding to this variant s u b f a m i l y is lower ia the al01 and a109 genomes or that this m',mber is the same for the five allotype genomes but the expression o f these genes is lower in a l 0 1 a n d a109 genomes due to some regulatory mechanisms.
RI~SUMI~ POLYMORPHISME Gt~NI~TIQUEDE LA RI~GIONVHa CHEZ LE LAPIN: L'ALLOTYPEa108
Nous d6crivons dans cet article un allotype VH du lapin jusqu'alors inconnu (a108) qui a 6t6 d6cel6 lors de l'6tude de la descendance d'un lapin de garenne (Oryctolagus cuniculus) originaire de Pile tunisienne de Zembra. Cet aUotype semble ~tre particulier/~ cette ile car il n'a pu ~tre retrouv6 chez d'autres populations de lapins sauvages (Espagne, Portugal et France). Les d6terminants allotypiques qui le caract6risent sont pr6sents sur le fragment Fd des cha/nes lourdes d'immunoglobulines, et il se comporte comme le produit d'un all61e au locus a. L'allotype a108 pr6sente une forte r6activit6 crois6e ave(: al et a107 et/~ un degr6 moindre avec les allotypes a101, a109 et a3. n a aussi 6t6 possible de mettre en 6vidence des d6terminants qui sont pr6sents de facon majoritaire sur lensmol6cules d'aUotypes al, a107 et a108 alors qu'on les retrouve sur une minorit6 des immunoglobulines d'allotypes al01 et a109. MolS-CLI~S: Allotypie, R6gion VHa, Immunoglobuline; Allotype nouveau a108, Lapin.
ACKNOWLEDGEMENTS
The authors wish to thank Dr T.J. Kindt for helpful discussion anf critical review of the manuscript. The excellent secretarial help of Ms M. Berson and the technical assistance of G. Severac are gratefully appreciated. This work was supported by Universit6 Pierre et Marie Curie, CNRS LA 359 and DRST (Tunisia)-CNRS Cooperation.
REFERENCES S. & TERNYNCK,T. (1967), Biologically active water-insoluble protein polymers. - I . Their use for isolation of antigen and antibodies. J. Biol. Chem. 242, 1651. BI~ZIN, C. & CAZENAVE, P.-A. (1975), La r6action crois6e entre le motif allotypique Aal des immunoglobulines du lapin et les anticorps dirig6s contre le motif allotypique Aa3: participation des variantes de la sp6cificit6 Aal /~ cette r6action crois6e. Immunochem. 12, 241. BP.EZIN, C., BEN~MAR, A., ROLAND, J. & CAZENAVE,P.-A. (1979), a and b allotypy in Oryctolagus and Lepus species. Ann. Immunol. (Inst. Pasteur), 130C, 167. Bpa~zrN, C. & CAZENAVE,P.-A. (1975), Allotypy of rabbit immunoglobulins: a fifth allele at the a locus. J. Immunol. 125, 59.
AVRAMEAS,
G E N E T I C P O L Y M O R P H I S M O F R A B B I T VHa R E G I O N
273
CAMBIASO, C.L., GOFFINET, A., VAERMAN, J.P. & HEREMANS, J.F. (1975), Glutaraldehyde-activated aminohexyl derivative of sepharose 4B, a new versatile immunoadsorbent. Immunochem., 12, 273. CAZENAVE, P.-A., Bg~ZXN, C. & ROLAND, J. (1974), An allotypic specificity presumably of the a series in rabbit immunoglobulins, different from al ; a2 and a3. Biochem. biophys. Res. Commun., 61, 664. CAZENAVE,P.-A. (1981), Immunoglobulin allotypes, in "Lymphocytic regulation by antibodies" (C. Bona & P.-A. Cazenave) John Willey & Sons, New York. FLEXSHMAN,J.B., PAIN, R.H. & PORTER,R.R. (1962), Reduction of gammaglobulins. Arch. Biochem. Biophys., 1, 174. GALLARDA,J.L., GLEASON,K.S. ,~ KNIGHT,K.L. (1985), Organization of rabbit immunoglobulin genes. - I. Structure and multiplicity of germline VH genes. J. Immunol., 135, 4222. G REENW'~O~~l~eC~uHmUa~nTg:;WhMfac&torGL<~gl~Js Se ( i l f 9 6 3 r ) a ' d ~ c ~ ~ ~ 12~!, 89, 114. HAOUAS, H., BENAMMAR-ELGAAIED, A. & CAZENAVE, P.-A. (1987), A "new allotype" of the a series in rabbit immunoglobulins (al07). MoL Immunol., 24, 247. KNlCHT, K.L., GILMAN-SACHS,A., FIELD, S.R. & DRAY, S. (1971), Ailotypic determinants on the Fab fragment of rabbit Aa-locus-negative IgGimmunoglobulin. J. Immunol. 106, 761. LANDUCCI-TOSl, S. & MACE, R.G. (1970), A method for typing rabbit sera for the A14 and AI5 allotypes with cross-linked antisera. J. Immunol., 105, 1046. LEVY, H.B. & SOBER,H.A. (1960), A simple chromatographic method for preparation of gammaglobulin. Proc. Soc. exp. Biol. (N.Y.), 103, 250. LOPEZ-MARTINEZ,N. (1977), Revision sistematica biostratigraphica de los lagomorpha (mammalia) del Neogenoy Cuaternario de Espana. Tesis Doctorat, Madrid. MACE, R.G. (1986), Rabbit immunoglobulin allotyoes, in "Handbook of immunology" (Weir, D.M., Herzenberg L.A., Blackwell, C.C. & Herzenberg, L.A. ) 4th Edition, Blackwell Scientific Publi., Edinburgh. MARGOLIES,M.N., CANNAN,E.L., KINDT, T.J. & FRASER,B. (1977), The structural basis of rabbit VH allotypes: serological stuaies on ai H chains with defined. A.A. sequence. J. Immunol., 119, 287. OUDIN, J. (1960a), A!lotype of rabbit serum proteins. -- I. Immmlochemic2J analysis leading to the individualization of seven main allotypes. J. exp. Med. 112, 107. OUDI~, J. (1960b), Allotypy of rabbit serum protein. -- !!. Re!ations~ps ber,veen various aUotypes: their common antigenic specificity, their distribution in a sample population, genetic implications. J. exp. Med. 112, 125. PIERLE, S.K. & KLINMAN,N.R. (1976), Allogenic carrier-specific enhancement of hapten specific secondary B-cell response. J. exp. Med., 144, 1254. PORTER, R.R. (1959), The hydrolysis of rabbit gammaglobulins and antibodies with cristalline papain. Biochem. J., 73, 119. ROLAND,J., BRI~ZIN,C. & CAZENAVE,P.-A. (1977), Cross-reaction of a minor variant of the al allotypic specificity with anti-a2 antibodies. Scand. J. ImmunoL, 6, 879. RotJx, K.H. (1981), A fourth heavy chain variable region subgroup, W, with 2 variants defined by an induced auto-antiserum in the rabbit. J. Immunoi. 127, 626. D ~ ,,utJx, K.H., GiLMAN-SACHS,A. & DRA¥, S. (1981), The identification of a VH subgroup allotypic specificity y30, which differenciates the y33 allele into two variants. Implications for rabbit VH gene organization and evolution. Mol. Immunol. 18, 359.
Res. l"m. m. .u .n .o .l .. . 1989, 140, 265-273
GENETIC POLYMORPHISM OF RABBIT Vna REGION: A NEW ALI.DTYPE, a108
H. Haouas (l) (,), A. E! Gaaied (2) and P.-A. Cazenave (l) (1) Unitd d'lmmunochimie Analytique, Ddpartement d'Immunologie, Institut Pasteur and LA CNRS 359 75724 Paris Cedex 15,
and (2) Laboratoire d'Immunogdndtique, Ddpartement des Sciences Biologiques, Facultd des Sciences de Tunis, Tunis
SUMMARY We report a new rabbit IgV H allotype, designated a108, which was detected following studies of the progeny of a wild rabbit (Oryctolagus cuniculus) from Zembra Island (Tunisia). This allotype seems to be specific of this island, since it has not been detected in other wild rabbit populations (in Spain, Portugal or France). Its determinants are located on the Fd fragment of the immunoglobulin (Ig) heavy chain and it behaves like the product of an allele at the a locus. Allotype a108 was strongly related serologically to al and a107, and to a lesser extent to al01, a109 and a3 allotypes. We also detected determinants shared among the al, a107, a108, al01 and a109 allotypes. These determinants were expressed by a large fraction of Ig molecules from rabbits of allotype al, a107 and a108 and by a very small fraction of Ig from rabbits of allotype al01 and a109. KEY-WORDS" Allotypy, VHa region, Immunoglobulin; New allotype a108, Rabbit. INTRODUCTION VH allotypes of rabbit immunoglobulins (Ig) reflect genetically controlled alternative structures of the Vri-gene segment 0VIage, 1986; Cazenave, 1981). Consequently, they are a valuable tool for understanding the evolution of VH genes in this species. Defined by specific serological reagents, the VH
Submitted April 3, 1989, accepted April 7, 1989. (*) To whomcorrespondence should be addressed.
266
H. H A O U A S E T A L .
allotypes have been subdivided into four subgroups, VHa, VHX, VHY and VHW, which are encoded by four VH-gene families: a, x, y, w (Mage, 1986; Knight et al., 1971 ; Roux, 1981, Roux et al., 1981). The VHa subgroup represents the majority of Ig, and the VHa-negative subgroups (VHx, VHY, VHW) only 10 to 30 070 of the total Ig. In domestic rabbits, three allelic forms of VHa subgroup: al, a2, a3, have been identified (Oudin, 1960a). Each of these allotypes appears to be a family of variants (Oudin, 196{)5) which differ in a few amino acid substitutions (Margolies et al., 1977). They appear to be encoded by closely linked genes (Oudin, 1960b) and are inherited together in the same proportion in sera of non-immunized rabbits (Oudin, 1960b; Roland et al, 1977). Studies of Ig heavy chains in wild rabbits, however, reveal that the polymorphism observed in the domestic rabbit represents only a fraction of the genetic poiymorphism of O. c~aniculus species. TMs is apparent because additional allotypes al00 to a109 have been detected in wild rabbit pop~ations in France, Spain, Portugal and Tunisia (Cazenave et al, 1974; Brezin and Cazenave, 1980; Brezin et al., 1979; Haouas et aL, 1987). In this paper, we describe the a108 ailotype found in the population of Zembra Island (Tunisia) and characterize its serological relatedness with different allotypes of the " a " series. In addition, we show that this allotype comprises a family of variants.
MATERIALS AND METHODS Purification of IgG, Fab and heavy and light chains.
IgG were purified from non-immune sera on DEAE cellulose (Levy and Sober, 1960). Fab was purified from IgG by papain hydrolysis (Porter, 1959). Heavy and light chains were separated by gel filtration after reduction and alkylation of IgG (Fleishman et al, 1962). Antisera and antibodies.
Anti-allotype antisera were prepared as previously described (Br~zin and Cazenave, 1975). Antibodies were isolated from antiallotypic antiserum on immunosorbent columns of IgG (Cambiaso et al., 1975). The antibodies adsorbed by passage through the Sepharose AH-4B immunosorbent column, were eluted with 0.2M glycine-HCl buffer pH 2.2 containing 0.SM NaCI. The eluate was immediately neutralized using 1M Tris buffer pH 9.
Ig = immunoglobulin.
[ RIA = radioimmunoassay.
GENETIC
POL YMORPHISM
OF RABBIT
Vfta R E G I O N
267
Fracfionation of IgG. Sepharose AH-4B coupled to antibody H620 (anti-al08 allotype serum) was incubated 2 h with al01 or a109 IgG (Cambiaso et al., 1975). The unboud fraction was removed by several washings with Tris (0.2M)/NaCI (0.SM) pH 8. The bound fraction was eluted by HCl/glycine/NaCl buffer. The same experiment was repeated until no protein was detected in the eluted fraction.
Radioimmunoassays (RIA). Reactions of the allotype with anti-allotype antibodies were studied by two RIA assays: liquid-phase RIA using insolubilized antisera (Landucci-Tosi and MaRe, 1970) and a solid-phase RIA using polyvinyl microtitre plates (Pierle aad Kliman, 1976). Antisera were insolubilized by polymerization with ethylchloroformate (Avrameas and Ternynck, 1967). IgG and Fab were 125I-labelled according to the Greenwood method (Greenwood et al., 1963).
Animals. The wild rabbits used were from Zembra Island (Tunisia). Domestic rabbits were Bouscat Giant.
RESULTS
Description of the a108 allotype. A m o n g the wild rabbits trapped on the Island of Zembra, one of them (LG896) carrying the VHa2 allotypic specificity was mated with a domestic doe homozygous a 3 / a 3 (fig. 1). None of the three offspring inherited the a2 aUotype f r o m the father; each exhibited the a3 +, a 2 - phenotype. One of
IN
L6896
I
®
I .-'H461 e~ i H747 FIo. I. - - Pedigree of LG 801 rabbit family.
C) = a2; A = a3; • = a108.
268
H. H A O U A S E T AL.
the litter (H461) was crossed with an a2/a3 domestic male. All offspring received the a3 allele from the buck. One of the males (H626) was randomly chosen and backcrossed with the H461 female. In seven progeny obtained, two produced sera that precipitated neither with anti-a3 sera nor with any antisera directed against the other allotypic specificities of the " a " series (al, a2, al00, al01, a106, a107, a109). This particular behaviour suggested that these individuals could be homozygous for an unknown allele at the a locus. Hence, an immune serum was prepared in an a3/a3 domestic rabbit against the IgG isolated from the serum of one of these individuals (H747). The av~iserum obtained was able to precipitate the immunizing IgG but was non-reactive with all known allotypes of the a series. Sera of all the family members were studied for their reactions with this antiserum by precipitation in liquid medium and RIA. Analyses si~owed that it behaved as an antiserum directed ag~nst an unknown aUotype of the a series, and we designated it a108 (fig. 1). The results summarized in figure 2 demonstrate that the a108 allotype is located on the heavy chain and Fab fragment.
.o//,o/
600
imm
in -m .elm .,C C: m
• IgG oH
50-
<>H reass
40-
• Fab AL
302010l
;
4.10-4
|
_/ i
2
4.t0"
;
A
l
4 nMIml
F I G . 2. - - Inhibition o f the binding o f 1251-labelled a108 + lgG to anti-al08 serum by IgG, Fab, heavy and light chains purified from H747 serum, and heavy chains reassociated with light chains isolated from b 4 / b 9 3 rabbit serum.
GENETIC POL YMORPHISM
OF RABBIT
Vh.a R E G I O N
~N
2v;,
TABLE I. - - Binding o f radiolabelled F a b to different antisera. Fab
Antisera
al
a2
a3
Anti-al (al01/al01) Anti-a2 (a3/a3) Anti-a3 (a2/a2) Anti-al00 (al01/al01) Anti-al01 (al/a3) Anti-al06 (al/a3) Anti-al07 (a2/a3) Anti-al08 (a3/a3) Anti-al09 (a3/a3)
82 . 75 58 .
70 -
62 . 98 47 . -
-
.
. . . .
. . .
allotype
(070)
al00 al01 a106 a107 a108 a109 71
-
.
. 58 91
. . .
76 . 55 89 . 84
. 76 -
82 . .
76 . 32 .
. . .
.
.
. 79 63
.
80
48
44 . 34 80
69 -
.
93
= binding below 25%.
a108 cross-reactivities.
The cross-reactivities of the various a allotypes were examined by binding 125I-labelled Fab from each of the allotypes to antiaUotypic antibodies against these various allotypes. As shown in table I, anti-al08 reacted with al and a107 allotypes, and conversely, anti-al, anti-al07 and anti-a3 reacted with a108 Fab. It is noteworthy that only a portion of the molecules (variants) of these allotypes were involved in several of these cross-reactivities. These results indicate that a108 molecules are comprised of two l=,opulations of
o am7 eat08 = al
//
/.
,, a!09 • aiOI oa2 • alO0 oa3
0
A
•
Dilution of inhibitor F [ o . 3. - -
Inhibition by different allotypic sera of the binding of radioiabeilcd a108 + Fab tt; purified homologous anti-allotypic antibodies.
270
H. H A O U A S E T AL.
molecules, one which reacts with anti-al07 (and/or anti-a3) and one which does not react with anti-al07 (or anti-a3). The a108 cross-reactivities have been studied in greater detail, using inhibition of the binding of al08+-labelled Fat) to homologous immune serum by different allotypes. As shown in figure 3, the reaction was inhibited by al, a107, al01 and a109 allotypes. These results show that the al and a107 allotypes carry most of the allot,~q~ic determinants recognized by the anti-al08 antiserum. However, the al and a107 allotypes were not precipitated by the anti-al08 antibodies. We concluded that these determinants have a different molecular distribution in the al, a107 and a108 allotypic families of variants. The results of additional experiments are in agreement with this interpretation. For instance, the a108 al!otype totally inhibited the binding of al+-and al07+-labelled Fab to homologous sera (data not shown). In another step, a108 cross-reactivities have also been studied by isolation of anti-al and anti-al07 antibodies capable of binding to al and a108 allotypes respectively. Inhibition of the binding of al08+-labeUed Fab to these isolated cross-reacting heterologous antiallotypic antibodies was studied and confirmed previous results, and, in addition, revealed that at least some of the allotypic determinants involved in al, a107 and a108 cross-reactivities are also present in a3, MOO, a106, al01 and a109 allotypic families.
. o ..-.
oa101la108+1 "~_
" :] i~/A~l/t
• all~
Aa3 .. ®al ,,.a. [ai08-I
.
Inhibitor (pg/ml) FIG. 4. - - Inhibition by different al!otypic sera o f the binding o f radiolabelled al + (A) or a107 + (B) Fab to anti-al08 antibodies purified on homologous allotype.
GENETIC POL YMORPHISM OF R A B B I T Vna REGION
271
TABLE II. - - I n h i b i t i o n by d i f f e r e n t allotypes of the binding of labelled F a b to homologous a n t i a l l o t y p i e sera. A l l o t y p e inlfibitor ( % ) Antisera
al
a2
a3
Anti-al08 (a3/a3) Anti-a1 (al01/al01) Anti-al07 (a3/a3)
99 89 84
- (*) -(**) -(**)
. .
al00
al01
a106
a107
a108
a109
-
96 . .
-
98 68 82
99 87 85
89 -
. .
. .
(*) inhibition below 20070 with a 10 -2 dilution of inhibitor serum. (**) Inhibition below 20070 with a 10-i dilution of inhibitor serum.
It was shown that the a101 and a109 allotypes did not bind to anti-al08 serum (table I) but were able to inhibit the binding of a108 allotype to homologous antiallotypic antibodies (table II) suggesting that only a small fraction of Ig molecules bearing the al01 and a109 determinants is responsible for the cross-reactivity with a108. As shown in figure 4, the a108 determinants involved in these cross-reactivities are those involved in the cross-leactivities with al and a107 allotypes.
DISCUSSION
The evidence presented in this paper indicates that the a108 allotype belongs to the a series. This allotype found on Zembra Island (Tunisia) has not been detected in other wild rabbit populations frcm France, Spain and Portugal (data not shown). However, the a108 aUotype has extensive cross-reactivity with allotypes having different geographical distributions: al and a3 (present in domestic rabbits and in several wild populations), al00 (present in France, Spain and Zembra), al01 (detected in France but not in other countries), a106 (particular to Portugal), a107 (present in Spain, Portugal and Zembra) and a109 (present in Spain and Portugal) (Haouas et al., 1987). These cross-reactivities reflect the complex evolution of the a allotypes. Understanding of the mechanisms of this evolution is clouded by the number of genes analysed which belong to the VH multigene family (Gallarda et al., 1985) and by the nature of the wild populations studied for which a "founders effect" is probably crucial, if one considers the natural history of the O. cuniculus species (Lopez-Martinez, 1977). It was shown that a major variant (or subfamily of variants) is present at a similar high concentration in the sera of al +, a107 + and a108 + rabbits. The same variant (or subfamily of variants) is detected, at a very low level
H. H A O U A S E T AL.
272
(1% of the allotype) in al01 + and a109 + sera. This observation suggests that either the n u m b e r o f VH genes corresponding to this variant s u b f a m i l y is lower ia the al01 and a109 genomes or that this m',mber is the same for the five allotype genomes but the expression o f these genes is lower in a l 0 1 a n d a109 genomes due to some regulatory mechanisms.
RI~SUMI~ POLYMORPHISME Gt~NI~TIQUEDE LA RI~GIONVHa CHEZ LE LAPIN: L'ALLOTYPEa108
Nous d6crivons dans cet article un allotype VH du lapin jusqu'alors inconnu (a108) qui a 6t6 d6cel6 lors de l'6tude de la descendance d'un lapin de garenne (Oryctolagus cuniculus) originaire de Pile tunisienne de Zembra. Cet aUotype semble ~tre particulier/~ cette ile car il n'a pu ~tre retrouv6 chez d'autres populations de lapins sauvages (Espagne, Portugal et France). Les d6terminants allotypiques qui le caract6risent sont pr6sents sur le fragment Fd des cha/nes lourdes d'immunoglobulines, et il se comporte comme le produit d'un all61e au locus a. L'allotype a108 pr6sente une forte r6activit6 crois6e ave(: al et a107 et/~ un degr6 moindre avec les allotypes a101, a109 et a3. n a aussi 6t6 possible de mettre en 6vidence des d6terminants qui sont pr6sents de facon majoritaire sur lensmol6cules d'aUotypes al, a107 et a108 alors qu'on les retrouve sur une minorit6 des immunoglobulines d'allotypes al01 et a109. MolS-CLI~S: Allotypie, R6gion VHa, Immunoglobuline; Allotype nouveau a108, Lapin.
ACKNOWLEDGEMENTS
The authors wish to thank Dr T.J. Kindt for helpful discussion anf critical review of the manuscript. The excellent secretarial help of Ms M. Berson and the technical assistance of G. Severac are gratefully appreciated. This work was supported by Universit6 Pierre et Marie Curie, CNRS LA 359 and DRST (Tunisia)-CNRS Cooperation.
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