Murine V kappa 21A isotype sequence: monoclonal antibody 50S10.1 specific for the group A streptococcal polysaccharide

Antibody 50S10.1 is a hybridoma-derived gamma 3 kappa antibody of BAB-14 mouse strain origin, with specificity for N-acetylglucosamine beta 1----3 linked to L-rhamnose, the immunodeterminant of the streptococcal Group A polysaccharide. The VL50S10.1 amino acid sequence is the fourth complete one reported with this specificity and the first fully determined V kappa 21A structure. Furthermore it is the first V kappa 21A isotype sequence derived from an antibody with known antigen specificity. The V kappa region of this and the previously described monoclonal anti-streptococcal Group A polysaccharide antibodies 7S34.1, 2S1.3 and 17S29.1 are compared, showing that in monoclonal antibody 50S10.1 a V kappa germline gene is expressed which is unrelated to those previously shown to be expressed in antibodies of this specificity. V kappa 50S10.1 increases the variability of known murine V kappa regions and confirms stretches of V kappa 21A sequences previously established.     

Murine V kappa 25 and V kappa 27 amino-acid sequences of C57B1/6 origin: monoclonal antibodies 17S29.1 and 22S25.1 specific for the group A-streptococcal polysaccharide

Antibodies 17S29.1 and 22S25.1 are monoclonal, hybridoma-derived gamma 3 kappa murine immunoglobulins with specificity for N-acetyl-glucosamine beta 1----3-linked to the L-rhamnose backbone structure, the immunodeterminant of the streptococcal Group A polysaccharide. The VL 17S29.1 amino-acid sequence is the third complete one reported from an antibody with this specificity, the second fully determined V kappa 25 structure and the first complete V kappa sequence of C57B1/6 origin derived from a carbohydrate-specific antibody. VL22S25.1 is a member of the V kappa 27 isotype of murine immunoglobulin VL regions. V kappa 17S29.1 and the determined part of the V kappa 22S25.1 sequence are compared to the previously described V kappa regions of streptococcal Group A polysaccharide-specific antibodies and to 12 selected partial and complete V kappa regions of antibodies with other specificities, predominantly to carbohydrate antigens. Both V kappa 17S29.1 and V kappa 22S25.1 increase the variability of known murine V kappa regions. They are the most homologous to the other V kappa regions derived from antibodies with streptococcal Group A polysaccharide specificity and share with them the amino-acid residue Arg74, so far characteristic for V kappa regions from antibodies with this specificity. The analysis of groups of independently expressed, highly homologous V kappa regions, namely V kappa 17S29.1 and V kappa 2S1.3 as one and V kappa 7S34.1 and V kappa 22S25.1 as a second group, offers the possibility of estimating the minimal number of V kappa germline genes involved in the immune response to the structurally defined streptococcal Group A polysaccharide antigen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein L from Peptostreptococcus magnus binds to the kappa light chain variable domain.

Protein L is an immunoglobulin light chain-binding protein expressed by some strains of the anaerobic bacterial species Peptostreptococcus magnus. The major variable region subgroups of human kappa and lambda light chains were tested for protein L binding; V kappa I, V kappa III, and V kappa IV bound protein L, whereas no binding occurred with proteins of the V kappa II subgroup or with any lambda light chain subgroups. Studies of the protein L binding capacity of naturally occurring VL fragments, and VL- and CL-related trypsin- and pepsin-derived peptides prepared from a kappa I light chain, localized the site of interaction to the VL domain. The affinity constant for the binding to an isolated V kappa I fragment was comparable to that for the native protein (Ka 0.9 x 10(9) M-1 and Ka 1.5 x 10(9) M-1, respectively). No binding occurred with CL-related fragments. Extensive reduction and alkylation of the V kappa fragment or the native kappa chain resulted in complete loss of protein L binding. Although it is possible, from comparative amino acid sequence data, to identify certain VL-framework region residues that account for the selective binding of protein L by kappa I, kappa III, and kappa IV proteins, our studies indicate that this interaction is essentially dependent upon the tertiary structural integrity of the kappa chain VL domain.     

Evolution of immunoglobulin kappa chain variable region genes in vertebrates

The major source of immunoglobulin diversity is variation in DNA sequence among multiple copies of variable region (V) genes of the heavy- and light-chain multigene families. In order to clarify the evolutionary pattern of the multigene family of immunoglobulin light kappa chain V region (V kappa) genes, phylogenetic analyses of V kappa genes from humans and other vertebrate species were conducted. The results obtained indicate that the V kappa genes so far sequenced can be grouped into three major monophyletic clusters, the cartilaginous fish, bony fish and amphibian, and mammalian clusters, and that the cartilaginous fish cluster first separated from the rest of the V kappa genes and then the remaining two clusters diverged. The mammalian V kappa genes can further be divided into 10 V kappa groups, 7 of which are present in the human genome. Human and mouse V kappa genes from different V kappa groups are intermingled rather than clustered on the chromosome, and there are a large number of pseudogenes scattered on the chromosome. This indicates that the chromosomal locations of V kappa genes have been shuffled many times by gene duplication, deletion, and transposition in the evolutionary process and that many genes have become nonfunctional during this process. This mode of evolution is consistent with the model of birth-and-death evolution rather than with the model of concerted evolution. An analysis of duplicate V kappa functional genes and pseudogenes in the human genome has indicated that pseudogenes evolve faster than functional genes but that the rate of nonsynonymous nucleotide substitution in the complementarity-determining regions of V kappa genes has been enhanced by positive Darwinian selection.      

Eukaryotic DNA topoisomerase I reaction is topology dependent.

The effects of supercoiling on the topoisomerization reaction by eukaryotic DNA topoisomerases I have been analyzed. The systems used were: DNA topoisomerase I from wheat germ, chicken erythrocyte and calf thymus on a 2.3 kb DNA fragment which encompasses the immunoglobulin kappa-light chain (L kappa) promoter of the mouse plasmacytoma MPC11; S. cerevisiae DNA topoisomerase I on a 2.2 kb DNA fragment from the same organism which encompasses the regulatory and the coding region of the ADH II gene; wheat germ DNA topoisomerase I on the plasmid pUC18. It was found in every system that lack of torsional stress prevents topoisomerization of the substrate. A simple regulatory model of DNA topoisomerase I function, based on topological considerations, is presented.     

Cloned MPC 11 myeloma cells express two kappa genes: a gene for a complete light chain and a gene for a constant region polypeptide.

Cloned MPC 11 mouse plasmacytoma cells synthesize a complete kappa light chain and also a kappa light chain constant region fragment. Partial amino terminal sequences of the in vitro forms of these two proteins have been determined. Both in vitro products contain typical light chain leaders; leaders are defined as the amino terminal sequences present on in vitro products but absent from the in vivo products found in living cells. The in vitro form of the MPC 11 complete light chain contains a leader sequence plus variable and constant region sequences. The in vitro form of the MPC 11 light chain constant region fragment contains a different leader sequence attached directly to a complete constant ragion sequence and has no variable region sequences. Thus the MPC 11 light chain fragment is not a degradation product of the MPC 11 complete light chain (or of any other complete light chain) and must be coded by a separate gene. The results reveal two unusual features of MPC 11 cells: first, expression of a unique variant light chain gene coding the light chain constant region fragment, and second, expression of two different kappa light chain genes (coding the complete light chain and the variant constant region fragment) in a single cell. In addition, evidence is provided that the in vitro forms of kappa light chains, three of which are presented here for the first time, include a minimum of three partially homologous but quite different leader sequences.     

Efficient amplification and direct sequencing of mouse variable regions from any immunoglobulin gene family.

We have designed two original sets of oligonucleotide primers hybridizing the relatively conserved motifs within the immunoglobulin signal sequences of each of the 15 heavy chain and 18 kappa light chain gene families. Comparison of these 5' primers with the immunoglobulin signal sequences referenced in the Kabat database suggests that these oligonucleotide primers should hybridize with 89.4% of the 428 mouse heavy chain signal sequences and with 91.8% of the 320 kappa light chain signal sequences with no mismatch. Following PCR amplification using the designed primers and direct sequencing of the amplified products, we obtained full-length variable sequences belonging to major (V(H)1, V(H)2, V(H)3, Vkappa1 and Vkappa21) but also small-sized (V(H)9, V(H)14, Vkappa2, Vkappa9A/9B, Vkappa12/13, Vkappa23 and Vkappa33/34) gene families, from nine murine monoclonal antibodies. This strategy could be a powerful tool for antibody sequence assessment whatever the V gene family before humanization of mouse monoclonal antibody or identification of paratope-derived peptides.     

Sequence of the full-length immunoglobulin kappa-chain of mouse myeloma MPC 11.

MPC 11 mouse myeloma cells synthesize two immunoglobulin kappa light chains, coded by two separate genes. One of these Kappa-chains has no variable region and is degraded intracellularly. The other is a full-length kappa-chain contaning both variable and constant regions: this chain is secreted, both by itself and combined with heavy chains in molecules of immunoglobulin G. This paper reports the amino acid sequence of the myeloma MPC 11 full-length kappa-chain. The chain is unusual in having 12 extra residues at its N-terminus when its sequence is aligned with those of other mouse kappa-chains; no other anomalies were found in its sequence.     

DNA sequence of the constant gene region of the mouse immunoglobulin kappa chain.

The constant (C; ref. 3) gene segment of the immunoglobulin kappa light chain and about 1 kb upstream as well as downstream of the segment have been sequenced. The sequences of the C gene segment itself and parts of the upstream region were determined both in liver and in myeloma T DNA clones derived from the same mouse inbred strain. The sequences were identical, i.e. no somatic mutations were detected. Two sites in the region not coding for protein are discussed as possible targets of aberrant variable (V) gene translocations. Doublet frequencies were calculated in the approx. 2500 bp of the C region sequence reported in this paper and in the approx. 3400 bp of two rearranged V gene regions.     

Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool

A common residue numbering scheme for all immunoglobulin variable domains (immunoglobulin light chain lambda (V(lambda)) and kappa (V(kappa)) variable domains, heavy chain variable domains (V(H)) and T-cell receptor alpha (V(alpha)), beta (V(beta)), gamma (V(gamma)) and delta (V(delta)) variable domains) has been devised. Based on the spatial alignment of known three-dimensional structures of immunoglobulin domains, it places the alignment gaps in a way that minimizes the average deviation from the averaged structure of the aligned domains. This residue numbering scheme was applied to the immunoglobulin variable domain structures in the PDB database to automate the extraction of information on structural variations in homologous positions of the different molecules. A number of methods are presented that allow the automated projection of information derived from individual structures or from the comparison of multi-structure alignments onto a graphical representation of the sequence alignment.     

Completion of the analysis of the primary structure of the variable domain of a homogeneous rabbit antibody to type III pneumococcal polysaccharide

The amino acid sequence between residues 70 and 116 of the V (variable) region of the H (heavy) chain derived from rabbit antibody BS-5, specific for type III pneumococcal polysaccharide, was determined. The sequence of this section of the H chain which includes the hypervariable residues 94 to about 112 was unique, although minor variant sequences present in the H chain preparation would not have been detected by the techniques used in this work. Taken together with the known sequences of the N-terminal 69 residues of H chain BS-5 (Jaton & Braun, 1972) and of the V region of the light chain (Jaton, 1974b), the data establish the complete sequence of the V domain of a rabbit immunoglobulin G. The V region of H chain BS-5 is compared with the basic sequences of the three human V region subgroups known to date, with one mouse H chain, and with guinea-pig pooled H chains. Even though chains from guinea pig and mouse clearly belong to the subgroup III of variability (V(HIII)), rabbit H chain BS-5 (allotypic variant a(1)) appears more closely related to the subgroup V(HII) than to the subgroups V(HIII) or V(HI). The homology between V(L) and V(H) regions of antibody BS-5 (28%) is not greater than that observed between the V(H) region of antibody BS-5 and the V(L) regions of different rabbit antibodies.     

Structural characterization of a cross-reactive idiotype shared by monoclonal antibodies specific for the human CD4 molecule.

A panel of mouse monoclonal anti-CD4 antibodies was characterized in terms of idiotypic expression by using specific anti-idiotypic antibody (anti-Id) reagents generated in rabbits immunized with anti-Leu3a, a monoclonal anti-CD4 which inhibits the human immunodeficiency virus (HIV) gp120 binding to CD4. Direct binding and competitive inhibition assays demonstrate that the majority of monoclonal anti-CD4 antibodies able to recognize CD4 epitopes overlapping the epitope recognized by anti-Leu3a expressed an antigen-combining site-related cross-reactive idiotype (IdX). Western blot analysis was used to demonstrate that this IdX is associated primarily with the light (L) chain of the monoclonal anti-CD4 antibodies. To further characterize the structural basis of the IdX, the nucleotide sequence of the variable region of the L kappa chain of anti-Leu3a was determined. Peptides corresponding to the first, second, and third complementarity determining regions (CDRs) of the L chain of anti-Leu3a were synthesized and used to immunize rabbits. All anti-peptide antisera recognized the immunizing peptide, the cognate anti-Leu3a molecule, and several other monoclonal anti-CD4 antibodies by direct binding assays. Western blot analysis utilizing the anti-CDR peptide reagents demonstrates that the reactivity to the monoclonal anti-CD4 antibodies was L chain-specific. The anti-Id generated by immunizing with the intact anti-Leu3a molecule failed to recognize the three L chain-derived CDR synthetic peptides, suggesting that the IdX requires the presence of the three-dimensional configuration of the L chain for its expression. The broad range of reactivity exhibited by the antipeptide antisera indicates that the majority of mouse monoclonal anti-CD4 antibodies characterized in this study utilize L chains encoded by a single germ line variable (V) region kappa (V kappa) chain gene or by V kappa genes that belong to the same gene family.     

Cloning and complete nucleotide sequence of mouse immunoglobulin gamma 1 chain gene.

The 6.6 kb DNA fragment coding for the immunoglobulin γ1 chain was cloned from newborn mouse DNA using λgtWES·λB as the EK2 vector. The complete nucleotide sequence (1823 bases) of the γ1 chain gene was determined. The cloned gene contained the entire constant region gene sequence as well as the poly(A) addition site, but not the variable region gene. The results indicate that the variable and constant region genes of immunoglobulin heavy chain are separated in newborn mouse DNA. The constant region genes of other gamma chains (that is, γ2a, γ2b and γ3) are not present in the cloned DNA fragment. The sequence demonstrates that the γ1 chain gene is interrupted by three intervening sequences at the junction of the domains and the hinge region, as previously shown in the γ2b and α chain genes and in the γ1 chain gene cloned from myeloma. The results suggest that the intervening sequence was introduced into the heavy chain gene before divergence of the heavy chain classes, and also support the hypothesis that the splicing mechanism has facilitated the evolution of eucaryotic genes by linking duplicated domains or prototype peptides not directly adjacent to one another. Comparison of the nucleotide sequence of the γ1 chain gene around the boundaries of the coding and intervening sequences with those of other mouse genes revealed extensive divergence, although short prevalent sequences of AG-GTCAG at the 5′ border of the intervening sequence and TCTGCAG-GC at the 3′ border were deduced. A limited homology of nucleotide sequences was found among domains and between the hinge region and the 5′ portion of the CH2 domain. Comparison of 3′ untranslated sequences from the γ1 and γ2b chain genes and the mouse major β-globin gene shows significant homology and a palindrome sequence surrounding the poly(A) addition site.     

Complete sequence of an immunoglobulin mRNA using specific priming and the dideoxynucleotide method of RNA sequencing.

The complete sequence of the mouse immunoglobulin kappa light chain MOPC 21 messenger RNA has been determined using a chain termination method and chemically synthesised deoxyoligonucleotides to initiate the synthesis of a DNA molecule complementary to the mRNA template. Five such oligonucleotide primers have been used for the sequence analysis of this messenger RNA. The approach is excellent for comparative studies of mouse k-chain mRNAs because they can be made on impure mRNA preparations. The MOPC 21 light chain mRNA is 943 nucleotides in length excluding the poly(A) region. An unexpected finding was that there are only three bases in the 5' non-coding region and its significance in terms of ribosome binding is discussed; 87 code for the precursor or leader sequence of the protein, 642 for the mature protein and 211 for the 3' non-coding region. The codons for the precursor region allows the previously undetermined amino acid sequence to be predicted. In common with other precursor regions a high proportion of the predicted amino acids are hydrophobic.     

Vectors for the expression of PCR-amplified immunoglobulin variable domains with human constant regions.

Cassette vectors have been constructed for mammalian expression of complete immunoglobulin heavy and light chain genes whose variable regions are produced by the polymerase chain reaction (PCR). The light and heavy chain vectors have promoter, leader, partial intron, enhancer and constant region segments within modified pSV2-gpt and pSV2-neo plasmids, respectively. Variable (V) regions are obtained by PCR using a two step process: 1) the V gene is amplified from genomic or cDNA, cloned into an intermediate vector and sequenced; 2) the first PCR product serves as the template for a second amplification in which restriction enzyme recognition sites and limited flanking intron sequence are added. The second PCR product is inserted into the expression vector, which is then transfected into mouse myeloma cells. These vectors contain human constant regions and may be used to express chimeric, humanized or human Ig genes. This report describes the design of these vectors and their application for the expression of chimeric 60.3, an anti-CD18 antibody.     

How immunoglobulin V kappa genes rearrange

Recombination at the immunoglobulin kappa light chain locus creates a complete variable region gene and its reciprocal product. The results presented here show that reciprocal products may be substrates for secondary recombination and that at least one V kappa group rearranges by inversion.     

Linkage of a 7S RNA sequence and kappa light chain genes in the mouse

A mouse 7S RNA cDNA plasmid clone was employed to identify and map DNA restriction fragment variants using recombinant inbred (RI) and congenic mouse strains. More than a dozen such restriction variants were identified and mapped to different regions of the mouse genome. One such variant, designated Rn7s-6, showed close linkage to the Ly-2,3-Igk-V (T lymphocyte antigens 2 and 3, kappa immunoglobulin variable region) cluster of markers on chromosome 6. No recombinants were detected among three of these markers in 59 RI strains. On the basis of these data, the Rn7s-6 sequence may be placed within 1.3 centimorgans of Ly-3 and one of the Igk-V-region markers, Igk-Efl. Two mouse stocks with previously identified crossovers within the Ly2,3-Igk-V region were used to sublocalize Rn7s-6. The results are consistent with the gene order (Ly-2, Ly-3)-(Rn7s-6, Igk-Efl)-Igk-Ef2. Several mouse plasmacytomas, known to have various parts of the kappa chain complex deleted, retain the Rn7s-6 sequence. The Rn7s-6 variant is a plus/minus variant; no sequence allelic to Rn7s-6 is found in inbred strains that share the Ly-3 ^a-Igk-Efl^a haplotype.     

Burkitt lymphoma cell line carrying a variant translocation creates new DNA at the breakpoint and violates the hierarchy of immunoglobulin gene rearrangement.

The Burkitt lymphoma cell line KK124, which contains a reciprocal t(8;22) translocation, was shown to have rearranged in a region 3' to the c-myc proto-oncogene on chromosome 8 and 5' to the lambda constant region on chromosome 22. The breakpoint was cloned and sequenced, revealing that c-myc and a portion of its 3' region abutted a complete lambda variable gene that had undergone V-J recombination. Since this cell line expresses kappa light chain, this lambda rearrangement violates the previously proposed hierarchy of immunoglobulin gene rearrangement. A novel duplication of normal chromosome 8 sequences was also found at the breakpoint. The first exon of c-myc and its flanking sequence from the translocated allele was sequenced and compared with a normal counterpart. Extensive mutation was found within the first exon in contrast to its 3' and 5' flanking regions. S1 nuclease analysis revealed that it was the translocated c-myc being expressed and that there was a promoter shift from P2 to P1. The detailed structural analysis of this cell line provides clues concerning mechanisms of chromosomal translocation and c-myc deregulation in Burkitt lymphomas.