Amino acid sequence of the light chain derived from a rabbit anti-p-azobenzoate antibody of restricted heterogeneity

A homozygous a2/a2 and b4/b4 rabbit has been hyperimmunized with Mirococcus lysodeiktiu. Poly(A)-containing RNA has been isolated from the spleen and translated in vitro, and translation products have been analyzed by NaDodSOd polyacrylamide gel electrophoresis. Double-stranded cDNA has been synthesized from poly(A)-containing RNA template and inserted in the Pst I endonuclease site of plasmid pBR322 by using the oligo(dC)-oligo(dG) tailing procedure. Tetracycline-resistant ampicillin-sensitive clones containing cDNA complementary to a sc light chain mRNA have been selected by differential screening and their ability to hybridize to a spleen mRNA having the same size as a mouse Klight chain mRNA. Two clones, pRk-15 and pRk-32, have been selected to determine the nucleotide sequence of the constant and 3' untranslated regions of X light chain mRNA, by the Maxam and Gilbert partial degradation method. Comparison of homologous regions ofmouse uc chain mRNA and b4 rabbit it chain mRNA reveals 61% homology in the constant region and 59% homology in the 3' untranslated region.Serological studies ofrabbit immunoglobulins have revealed an important genetic polymorphism called allotypy (1, 2). Structures responsible for allotypic specificities have been located on constant (C) and variable (V) regions of heavy chains and on the C region oflight (L) chains (for a review, see ref.3). Genetic studies on the inheritance of allotypes have shown that these polymorphic forms segregate in a Mendelian fashion, implying that they are encoded by allelic structural genes (4).Four allotypic forms ofthe domestic rabbit K chain have been characterized (5, 6). They are designated by the symbols b4, b5, b6, and b9. The sequences ofthe CK region of b4 and b9 chains exhibit a surprising divergence; they differ by 35% of their amino acid sequences, and nine two-base substitutions and three gaps are found (7,8). Furthermore, serological analysis has shown that one heterozygous b4/b5 rabbit can express three allotypes of the b series (b4, b5, b6) (9). The extensive divergence of allelic forms of rabbit K chains and the occurrence of latent allotypes have led to the hypothesis that b allotypes in the rabbit are encoded by multiple closely linked genes regulated by a polymorphic control mechanism (8,10). Studying, at the DNA level, the gene segments coding for the rabbit b allotypes presents two main difficulties: (i) there is no rabbit myeloma cell line; (i) the extensive divergence between rabbit and mouse C sequence (44% homology at the amino acid level) makes uncertain the possibility of screening a rabbit cDNA library with a cloned mouse CK probe.Starting from hyperimmunized rabbit spleen, we have constructed cDNA clones complementary to a rabbit K chain mRNA. The sequence of the constant and 3' untranslated regions of a rabbit b4 K chain mRNA has been determined and compared to corresponding mouse sequences. MATERIALS AND METHODSEnzymes and Chemicals. Terminal deoxynucleotidyl transferase, phage T4 polynucleotide kinase, and rest...

Section: Discussionmentioning

confidence: 99%

Nucleotide sequence of constant and 3' untranslated regions of a kappa immunoglobulin light chain mRNA of a homozygous b4 rabbit.

Heidmann¹,

Auffray²,

Cazenave³

et al. 1981

“…Prolonged and repetitive immunization of rabbits with bacterial vaccines (6,7) results in the frequent production of monoclonal antibodies that are suitable for determination of primary sequence. Occasionally responses to hapten-protein conjugates are also monoclonal (8). The amino-acid sequences of the light chain variable region (VL) in rabbit antibody to p-azobenzoate (8), group C streptococcus (9), and two antibodies specific for Type III pneumococcal polysaccharide (S3) (10,11) have been described.…”

mentioning

confidence: 99%

“…Occasionally responses to hapten-protein conjugates are also monoclonal (8). The amino-acid sequences of the light chain variable region (VL) in rabbit antibody to p-azobenzoate (8), group C streptococcus (9), and two antibodies specific for Type III pneumococcal polysaccharide (S3) (10,11) have been described. We report here complete VL amino-acid sequences of a Type VIII pneumococcal polysaccharide (S8)-specific antibody, two S3-specific antibodies, as well as partial VL sequences for three additional S8 and one additional S3 antibody and an antibody specific for Mficrococcus lysodeikticus (Al.…”

mentioning

confidence: 99%

Diversity of light chain variable region sequences among rabbit antibodies elicited by the same antigens.

Margolies¹,

Cannon²,

Strosberg³

et al. 1975

We report the complete variable region sequences of three homogeneous rabbit antibody light chains and the partial sequences of five others. When these are compared to other published rabbit light chain sequences, two regions of markedly increased variability are revealed, which are homologous in position to the first and third hypervariable regions of murine and human myeloma light chains. In addition, there is increased variability among the first three residues at the aminoterminal end. A hypervariable region homologous to that identified at positions 50 to 56 in myeloma light chains is not present in these rabbit antibody light chains. The available three-dimensional models of Fab fragments based on x-ray crystallography indicate that neither the amino-terminal portion of the light chain nor the region homologous to positions 50 to 56 forms a part of the combining site.Comparison of the hypervariable regions among six light chains from antibodies to Type III pneumococcal polysaccharide and among four from antibodies to Type VIII pneumococcal polysaccharide suggests that a large number of different sequences may be found in antibodies specific for these relatively simple antigens.Certain residues outside of the hypervariable regions are invariant in the rabbit light chains and correspond to residues that are required for proper chain folding in human and murine myeloma light chains, indicating that the general conformation of myeloma light chains is the same as that of light chains of elicited antibodies.An examination of amino-acid sequences from many paraprotein light chains (L chains) (1) and heavy chains (H chains) (2, 3, 36) has revealed segments of "hypervariability" within the variable region, which are thought to include residues involved in antigen binding. X-ray crystallographic studies of hapten-binding human (4) and murine (5)

“…2). The sequence is remarkably different from that ofthe A chain (13 substitutions in 28 residue positions) and also distinct from the sequences of b4, b6, and b9 K chains (7)(8)(9) Kbas 6038…”

Section: Resultsmentioning

confidence: 99%

“…Extensive chemical differences in the constant (C) regions of b4, b5, b6, and b9 molecules have been reported (7)(8)(9) and are thought to reflect the expression of the antigenic determinants of the b allotypes (10). In standard domestic rabbits, A-type L chains are the minor component of the immunoglobulin L chain pool (11).…”

mentioning

confidence: 99%

BASILEA rabbits express two types of immunoglobulin light chains: lambda and kappa-like.

García¹,

Brandt²,

Benammar³

et al. 1982

In the variant rabbit strain BASILEA, immunoglobulins G were shown to contain two distinct populations of IgG molecules whose light (L) chains belonged to the known A isotype and to a new K-like type. These two L chains differed from each other by electrophoretic, chemical, and antigenic properties. The K-like L chain fraction showed (i) an acid-labile Asp-Pro bond at the end of the joining region and (ii) a tryptic peptide, whose amino acid sequence ofthe NH2-terminal 15 residues was identical to the homologous constant (C) region sequence of b9 K chain with the exception of the residue in position 70, which is asparagine in the K-like chain instead of the characteristic half-cystine residue in all L chains of KB type expressing b4, b5, b6, or b9 allotypes. Serological and chemical studies have demonstrated to date the existence of five polymorphic forms of the domestic rabbit K light (L) chain allotypes designated b4, b5, b6, b9, and b4var (1-6). Extensive chemical differences in the constant (C) regions of b4, b5, b6, and b9 molecules have been reported (7-9) and are thought to reflect the expression of the antigenic determinants of the b allotypes (10). In standard domestic rabbits, A-type L chains are the minor component of the immunoglobulin L chain pool (11). However, this situation is reversed in a variant rabbit strain designated BASILEA, which was developed and maintained by Kelus and Weiss (12). Homozygous BASILEA rabbits are characterized by the lack ofK chains bearing b4, b5, b6, or b9 allotypic markers, which are substituted by L chains of the A type bearing C7 and C21 allotypic specificities (12-14). The primary structure of the constant region ofA L chains derived from an antibody IgG raised in BASILEA rabbits was recently completed and showed extensive homology with A chains of other species (14,15