Multiple sequences related to a constant-region kappa light chain gene in the rabbit genome

Four allelic genes control kappa light chain allotypes in the rabbit. Amino acid sequence studies have revealed an extensive divergence (22%--33%) in the alternative forms of the kappa constant region (b4, b5, b6 and b9). Furthermore, independent studies have shown that a rabbit could express a wrong allotype. To assess the hypothesis that b allotypes are encoded by duplicated genes with a polymorphic control mechanism, we have analyzed the DNAs of four different homozygous rabbits using the Southern blot hybridization technique, with a cloned b4 C kappa probe. DNAs of individual b4, b5, b6 and b9 rabbits were cleaved with Eco RI, Kpn I and Pst I restriction endonucleases. Comparative analysis of restriction patterns shows that all four rabbit DNAs contain multiple DNA fragments hybridizing with the probe under low- and high-stringency washing conditions. In addition, each restriction pattern is distinct, suggesting that the C kappa genes are organized differently in animals expressing different allotypes.     

Allotypes of the constant region of the rabbit T cell receptor beta-2 chain

Our laboratory previously reported that there was restriction fragment length polymorphism of TCR C beta genes in rabbits. EcoRI digests of DNA from different rabbits gave fragments of 9 and 6 kb (C beta a) or 14 and 6 kb (C beta b) that hybridized to a C beta cDNA probe. We also reported that the 9- and 14-kb types segregated as Mendelian traits and that there were allotypic differences in the first exon of the C beta 1 genes of C beta a and C beta b animals. Here we report the DNA sequence of the C beta 2 gene present in the 6-kb EcoRI fragment from a C beta b animal and compare the exon sequences with that of a cDNA from a C beta a animal. We find replacement changes in the first and third exons that probably represent allotypic forms of the rabbit C beta 2 gene. The genomic DNA 5' of exon 1 of both beta 1 and beta 2 contain alternating purine/pyrimidine repeat sequences. The genomic C beta 2 has an open reading frame of 69 amino acids in frame with exon 1 similar to a longer one previously found 5' of exon 1 of C beta 1. Further 5' of this region, rabbit C beta 1 and C beta 2 DNA sequences are only about 66% similar. Both the C beta 1 and C beta 2 sequences have two chi sequences; one in exon 1 with a perfect match and one in the intron downstream of exon 1 with one mismatch. Alternating purine/pyrimidine repeats and chi sequences found in rabbit C beta 1 and C beta 2 genes may have contributed to process(es) of gene duplication and/or conversion.     

Genomic DNA encoding rabbit T cell receptor beta-chains: isotypes and allotypes of C beta

We have discovered sequence differences in DNA encoding the first exon of rabbit T cell receptor beta-chains from unrelated rabbits that probably reflect allelic C beta 1 allotypes. Rabbit I was from a colony bred to maintain the K1-expression mutation Basilea, and rabbit II was from a colony bred to maintain the K1b9 allotype. Genomic DNA from rabbits I and II also exhibit restriction fragment length polymorphism of C beta on Southern blots. In addition, several different restriction enzyme digests of DNA from rabbit I give three bands, whereas DNA from rabbit II gives two when probed with C beta. An approximately 14-kb cloned genomic DNA fragment from rabbit I has two copies of C beta exon 1 and a 6-kb fragment has a third copy, suggesting that rabbit I has three different C beta genes. The DNA sequence of a germ-line genomic DNA fragment encoding the first exon of the beta-chain constant region from rabbit I also has an open reading frame encoding 140 amino acids immediately 5' of the C beta sequence. A corresponding sequence had previously been found in a cDNA clone from the second rabbit (rabbit II).     

Identification of rabbit genomic Ig-VH pseudogenes that could serve as donor sequences for latent allotype expression

Synthetic DNA oligomers specific for the VHa allotypes of rabbit Ig genes have been used to identify latent allotypic sequences in homozygous a1 and a2 rabbits. Two Ig VH pseudogenes containing latent a3 regions have been cloned from the genome of a homozygous a2 rabbit. Analysis of the regions associated with allotype expression indicates that these two pseudogenes contain VHa- sequences in framework region 1 (FR1) and VHa3 sequences in FR3. One gene has undergone an unusual rearrangement with a third VH gene, deleting their intervening sequences and recombining in FR3 with sequences 5' to the leader exon. Our results demonstrate the presence of latent VH sequences in the genomic DNA of normal rabbits and suggest that a mechanism such as gene conversion is responsible for expression of genetically-unexpected Ig VH genes.     

Expression of group b light chain allotypes in cottontail rabbits.

Group b allotypic determinants (b4, b5, b6, and b9) were detected on cottontail IgG by several assays including immunodiffusion, radioimmune binding, and inhibition of radiobinding. The results indicate that cottontail IgG possess some but not all of the subspecificities present on domestic rabbit IgG. Several cottontail rabbits exhibited three of the four possible allotypic markers. In these instances, however, only two populations of IgG molecules (i.e., phenogroups) could be detected, each bearing one, two, or three of the allotypes. Five separate and distinctive phenogroups were identified. The results suggest that the phenogroups represent products of multiple allelic genes for the constant region of cottontail rabbit kappa light chain.     

Organization of rabbit immunoglobulin genes. I. Structure and multiplicity of germ-line VH genes

Two rabbit germ-line VH gene segments have been isolated from a recombinant phage DNA library. Nucleotide sequence analysis indicates that both of the genes share structural and regulatory features common to mouse and human VH genes, although one appears to be a pseudogene. Comparison of the protein sequences encoded by these genes to the protein sequences of rabbit immunoglobulin V regions indicates that both genes encode VH a-negative-like molecules. Quantitative genomic blot analysis with a VH probe capable of recognizing most, if not all, germ-line VH genes indicates that there are approximately 100 VH genes in the haploid genome of rabbits. The average spacing between the germ-line VH genes was determined to be approximately 6 kb. The molecular basis for the allelic inheritance of rabbit VH allotypes is discussed in view of the structural organization of germ-line VH genes.     

Molecular basis of the allelic inheritance of rabbit immunoglobulin VH allotypes: implications for the generation of antibody diversity

Rabbits are unique in that their immunoglobulin VH regions bear allotypic markers encoded by allelic genes. The presence of these markers on most serum immunoglobulins is difficult to explain, as the germline contains several hundred VH genes. We cloned VH genes from normal rabbits of the VHa allotypes a1, a2, and a3 and from a mutant a2 rabbit, Alicia, which expresses almost no a2 allotype. The D-proximal VH gene VH1 of normal rabbits encoded prototype a1, a2, or a3 allotype VH regions in a1, a2, or a3 rabbits, respectively; VH1 was shown to be preferentially utilized in leukemic rabbit B cells. This VH1 gene was deleted from the germline of the Alicia rabbit. These data suggest that the allelic inheritance of a allotypes results from preferential utilization of VH1 in VDJ rearrangements. We suggest that antibody diversity in rabbit primarily results from somatic hypermutation and gene conversion.     

Organization and polymorphism of rabbit immunoglobulin heavy chain genes

Germline genes encoding C mu, C gamma, C alpha, and C epsilon heavy chains of rabbit immunoglobulins have been isolated from recombinant phage and cosmid libraries. The JH, C mu, C gamma, and C epsilon are found in a 5'-JH-C mu-C gamma-C epsilon-3' orientation on a 90kb stretch of DNA. Four C alpha genes have been cloned and presumably reside 3' to the other CH genes. Southern blot analysis of rabbit sperm DNA indicates that the rabbit genome contains a single C gamma gene, one C mu gene, and as many as 10 C alpha genes. Restriction site polymorphism is found for C mu, C gamma, and C alpha genes of rabbits of various heavy chain haplotypes. The organization of the rabbit CH genes differs from that of mouse and human CH genes in that the rabbit has multiple C alpha genes, whereas mouse and human have one or two C alpha genes, respectively. In addition, mouse and human have four C gamma genes, whereas rabbit has only one C gamma gene. The presence of a single C gamma gene indicates that at least in the rabbits examined, no germline gene encoding latent or unexpected, C gamma allotypes is present. The genetic control of the expression of latent C gamma allotypes is discussed.     

cDNA clones encoding rabbit immunoglobulin lambda chains. Evidence for length variation of the third hypervariable region and for a novel constant region.

Five cDNA clones designated pDH2, pDH8, pDH9, pDH31 and pDH101 encoding rabbit immunoglobulin lambda light chain sequences have been characterized. Comparison of the V lambda sequences suggests that, in addition to an increased divergence in all of the complementarity-determining regions (CDRs), variable-region diversity is amplified by the length heterogeneity of the CDR3, at the V lambda-J lambda junction. An insertion of four codons at positions 48a-d has been noted in three cDNA sequences. This insert, not found in lambda nor kappa light chains of other species, has a variable sequence, suggesting its possible implication in expanding variability of the CDR2. One of the cDNA clones was shown to encode a novel C lambda region which differs by four amino acid substitutions from the C lambda region common to all the other clones. Thus, the rabbit can use two different C lambda genes, which might correlate with the expression of the two known allotypes of lambda chains, C7 and C21. Southern blotting experiments indicate a small number of germ-line V lambda genes and the cDNA nucleotide sequence data reported here suggest that several of these genes can be expressed. The possibility of at least two V-J-C gene clusters is discussed.     

Rabbit immune repertoires as sources for therapeutic monoclonal antibodies: the impact of kappa allotype-correlated variation in cysteine content on antibody libraries selected by phage display

The rabbit immune repertoire has long been a rich source of diagnostic polyclonal antibodies. Now it also holds great promise as a source of therapeutic monoclonal antibodies. On the basis of phage display technology, we recently reported the first humanization of a rabbit monoclonal antibody. The allotypic diversity of rabbit immunoglobulins prompted us to compare different rabbit immune repertoires for the generation and humanization of monoclonal antibodies that bind with strong affinity to antigens involved in tumor angiogenesis. In particular, we evaluated the diversity of unselected and selected chimeric rabbit/human Fab libraries that were derived from different kappa light chain allotypes. Most rabbit light chains have an extra disulfide bridge that links the variable and constant domains in addition to the two intrachain disulfide bridges shared with mouse and human kappa light chains. Here we evaluate the impact of this increased disulfide bridge complexity on the generation and selection of chimeric rabbit/human Fab libraries. We demonstrate that rabbits with mutant bas and wild-type parental b9 allotypes are excellent sources for therapeutic monoclonal antibodies. Featured among the selected clones with b9 allotype is a rabbit/human Fab that binds with a dissociation constant of 1nM to both human and mouse Tie-2, which will facilitate its evaluation in mouse models of human cancer. Examination of 228 new rabbit antibody sequences allowed for a comprehensive comparison of the LCDR3 and HCDR3 length diversity in rabbits. This study revealed that rabbits exhibit an HCDR3 length distribution more closely related to human antibodies than mouse antibodies.     

Partial amino acid sequence of a rabbit immunoglobulin light chain of allotype b5

The amino acid sequence of a rabbit immunoglobulin light chain of allotype b5 has been nearly completed. A comparison of its structure with that of light chains of allotypes b4, b6, and b9 confirms that the constant regions of these various kappa chains differ by 20-35%. The substitutions are clustered in parts of the second half of the chain, and the b5 form bears more resemblance to the b6 chain than to any other, in good agreement with previous serological data. The analysis of the variable region reveals the existence of certain allotype-associated residues which have also been reported in other b5 chains, but not in proteins of the other allotypes. An examination of the rabbit light chain sequences between positions 96 and 107 suggests that this portion of the chain may be encoded separately by a joining "J" DNA segment, as has been described previously for murine and human immunoglobulins. In the rabbit, however, these J kappa regions appear to differ from one allotype to another. Together with the extensive variations of the constant regions, these data suggest that the rabbit kappa gene organization more closely resembles the murine gamma system (four different C gamma genes each flanked by its J segment) than the murine kappa system (a single C kappa gene).     

Two V kappa germ-line genes related to the GAT idiotypic network (Ab1 and Ab3/Ab1') account for the major subfamilies of the mouse V kappa-1 variability subgroup

V kappa Ig germ-line genes have been isolated from recombinant clones prepared in separate libraries constructed from adult BALB/c liver DNA. Three different clones that strongly hybridized with a V kappa-GAT-specific probe were completely characterized and sequenced. All three genes exhibited common characteristic features in their sequences encompassing the 5' to the 3' noncoding region, with coding sections 95% homologous. A comparison with other V kappa genes shows that the size of the first intron is variability subgroup specific. Moreover, a direct correlation exists between the size of this intron and the entire length of the coding region. Nucleotide sequences of these genes were compared with V kappa chains expressed at the Ab1 and Ab1' levels of the GAT idiotypic network: Ag----Ab1----Ab2----Ab3 (Ab1'). K1A5 and K5.1 genes account for V kappa chains in Ab1 and Ab1' hybridomas, respectively. The high conservation of Ab1' sequences in light chain was also recently reported for the heavy chains, suggesting that immunization with Ab2 (anti-idiotypic) antibodies preferentially stimulates the direct expression of germ-line genes. K5.1 and K1A5 genes belong to the V kappa-1 variability subgroup and encode, without any amino acid substitution, V kappa domain in myeloma TEPC 105 and MOPC 467, which are V kappa-1A and V kappa-1C subgroup prototypes, respectively. These genes are extensively used in different mouse strains and in a number of antibodies of discrete specificities, such as anti-GAT, anti-DNP, anti-flagellin, anti-phosphorylcholine, anti-digoxin, anti-phenyloxazolone, and anti-DNA.     

Interallelic and intergenic conversion events could induce differential evolution of the two rabbit immunoglobulin kappa light chain genes.

Contrary to the situation in humans or mice, where the constant region (C) of the Immunoglobulin (Ig) kappa (kappa) light chain is encoded by a single gene, the rabbit possesses two C kappa genes: C kappa 1 and C kappa 2. However, in domestic rabbits, the vast majority of the immunoglobulins have a light chain of the kappa 1 isotype, which is expressed under four complex, highly divergent allelic forms: b4, b5, b6 and b9. In previous papers, we have shown that this high level of divergence was due, at least partly, to conversion events of the kappa 1 by the kappa 2 locus. Up to now, little was known about the evolution of the C kappa 2 gene. Here, we report sequences of the C kappa 2 genes in three different haplotypes, and show that, in contrast to the situation in the kappa 1 locus, the three analysed C kappa 2 alleles are identical (or only differing by one silent substitution). This suggests that intergenic conversion, which introduced most of the divergence in the kappa 1 locus, is not reciprocal and is unidirectional from kappa 2 towards kappa 1. To explain the small number of silent substitutions in the C kappa 2 gene and its remarkable conservation, we propose an extended model of multigenic family evolution, which postulates that gene conversion events occur between linked genes as well as between alleles.     

Surface immunoglobulin on rabbit lymphoid cells. III. Double expression and separate endocytosis of surface immunoglobulin allotypes on heterozygous lymphocytes demonstrated by immunoelectron microscopic labeling.

The expression of immunoglobulin b locus (k chain) allotypes on the surface of rabbit peripheral blood lymphocytes (PBL's) is examined using an indirect double immunoelectron microscopic labeling technique. Ferritin and whelk hemocyanin individually conjugated to allotypically specific IgG are used as ultrastructurally identifiable molecular markers. These indicators are coupled to lymphocyte surface immunoglobulin (Ig) allotypic determinants by an antiallotype antibody linkage. Human red blood cells, conjugated with IgG of a specific allotype and used as test cells, demonstrate the absolute specificity and high efficiency of the ultrastructural labeling technique. Specific labeling on rabbit PBL's shows that 65–75% of the cells are positive for surface Ig. Lymphocytes from homozygous donors (b4b4 or b6b6) are labeled specifically with only the appropriate allotypic labeling system. Thirty-three percent of the PBL's from heterozygous donors (b4b6) express both allotypes (allelic inclusion) on the cell surface; the remaining proportion of Ig-bearing cells have only one detectable allotype present (allelic exclusion). We conclude that approximately 50% of the Ig-bearing PBL's demonstrate allelic inclusion for the b locus allotypes. On allelically included heterozygous lymphocytes, both allotypic determinants can undergo specific endocytosis. Endocytosis of one allotype on heterozygous cells can be induced by stimulation with antiallotypic serum without affecting the surface appearance of the other allelic marker (separate endocytosis).     

The sequences of rabbit kappa light chains of b4 and b5 allotypes differ more in their constant regions than in their 3'' untranslated regions.

We report the sequence of a cDNA clone encoding the entire variable and constant regions of a rabbit kappa light chain of b5 allotype. The deduced amino acid sequence of the variable region (positions 1-95) is 86% homologous to that of a b4 light chain protein [BS-1) (1) but the b4 and b5 constant regions are only 74% homologous. Comparison of this DNA sequence to that of a cDNA clone encoding a b4 constant region shows that the kappa allotypes b4 and b5 have diverged significantly more in their coding region than in the 3' untranslated regions (86% vs 96% nucleotide sequence homologies). This implies either a function for the 3' untranslated region with evolutionary pressures to conserve or an accelerated divergence of the coding regions.     

Identification and genetic control of two rabbit high-density lipoprotein allotypes

Rabbits were immunized with high-density lipoprotein (HDL) isolated from the serum of other rabbits by ultracentrifugation and gel filtration. Two different precipitating antibodies were elicited which distinguished two antigenically different genetic variants, i.e., allotypes, of HDL. The allotypes were identified as HDL based on the observation that on immunoelectrophoresis the antigen-antibody precipitate formed by the reaction of each of the antiallotype antisera with electrophoresed rabbit serum appeared electrophoretically in the α₁ region and reacted with Sudan black and with β-naphthyl acetate. In addition, the precipitin bands formed by the reaction of each antiallotype antiserum with a normal rabbit serum coalesced with the precipitin band formed by the reaction of goat anti-HDL with the same normal rabbit serum. The inheritance of the two allotypes is controlled by a pair of allelic genes, as shown by genetic studies of 312 progeny from 83 rabbit families. This HDL locus, designatedLhj, was shown not to be linked to theLpq locus of low-density lipoprotein nor to any of five other loci controlling allotypic specificities of different rabbit serum proteins. The use of these allotypes for studying the structure and biosynthesis of HDL is discussed. This study was supported by Research Grant PHS AI-07043 (Dr. S. Dray) and PHS AI-09241 from the National Institutes of Allergy and Infectious Diseases. One of us (K. L. K.) is the recipient of National Institutes of Health Career Development Award (AI-28687).     

Human immunoglobulin heavy chain A2 gene allotype determination by restriction fragment length polymorphism.

The human immunoglobulin heavy chain alpha 2 genes have two allelic forms or allotypes called A2m(1) and A2m(2). Previously, these allotypic markers have only been distinguishable by serology. Studies of the alpha 2 genes, however, show that it is possible to differentiate between the allotypes by restriction enzyme site polymorphisms, both in the protein coding regions and in flanking regions. These polymorphic sites have been used to determine the alpha 2 allotypes of several human DNAs.     

Genes encoding Xenopus laevis Ig L chains. Implications for the evolution of kappa and lambda chains.

Xenopus laevis Ig contain two distinct types of L chains, designated rho or L1 and sigma or L2. We have analyzed Xenopus genomic DNA by Southern blotting with cDNA probes specific for L1 V and C regions. Many fragments hybridized to the V probe, but only one or two fragments hybridized to the C probe. Corresponding C, J, and V gene segments were identified on clones isolated from a genomic library prepared from the same DNA. One clone contains a C gene segment separated from a J gene segment by an intron of 3.4 kb. The J and C gene segments are nearly identical in sequence to cDNA clones analyzed previously. The C segment is somewhat more similar and the J segment considerably more similar in sequence to the corresponding segments of mammalian kappa chains than to those of mammalian lambda chains. Upstream of the J segment is a typical recombination signal sequence with a spacer of 23 bp, as in J kappa. A second clone from the library contains four V gene segments, separated by 2.1 to 3.6 kb. Two of these, V1 and V3, have the expected structural and regulatory features of V genes, and are very similar in sequence to each other and to mammalian V kappa. A third gene segment, V2, resembles V1 and V3 in its coding region and nearby 5'-flanking region, but diverges in sequence 5' to position -95 with loss of the octamer promoter element. The fourth V-like segment is similar to the others at the 3'-end, but upstream of codon 64 bears no resemblance in sequence to any Ig V region. All four V segments have typical recombination signal sequences with 12-bp spacers at their 3'-ends, as in V kappa. Taken together, the data suggest that Xenopus L1 L chain genes are members of the kappa gene family.