Immunoglobulin kappa-genes. Cloning, hybridization and structural analysis

The principal problems in molecular and genetic immunology to be resolved are the structure of Ig-genes and the regulation of their expression. The isolation of mRNA for light and heavy Ig-chains would be a first step along this line. A combination of two approaches may be the best strategy in mRNA preparation. Affinity purification allows one to obtain pure mRNA in a one-step procedure whereas immunoprecipitation makes it possible to prepare mRNAs for both heavy and light immunoglobulin chains in considerable amounts. In the series of experiments, conditions for synthesis of long and short cDNA chains were elaborated and the clone containing the fragment of kappa-chain gene was isolated and characterized. This clone was used as a probe for hybridization with genomic DNA from myeloma and hybridoma cells. It was shown that both allelic genes on homologous chromosomes were rearranged in myeloma MOPC21 cells. The original cell line of hybridoma PTF02 contains the embryonic gene as well as the differentiated genes. However, only differentiated genes can be detected in a similar experiment conducted with the same hybridoma after passage on mice. In conclusion, the coordination of homologous and heterologous chromosome expression in B cells in discussed in terms of the feed-back control.     

Arrangement of lambda light chain genes in mutant clones of the MOPC 315 mouse myeloma cells

The synthesis of lambda light chains and the arrangement of the lambda-chain genes was examined in cells of the mouse myeloma MOPC 315, which is an alpha lambda 2 producer, and in several mutants derived from it. The mutants produce lambda 2 chains only (MOPC 315.26, MOPC 315.34, and MOPC 315.37) or fail to produce alpha and lambda 2 chains (MOPC 315.25 and MOPC 315.36). Messenger RNA from the lambda 2 chain-producing cells directed the synthesis of a lambda 2 chain precursor and a fragment of the lambda 1 chain (lambda 1 F) in a wheat embryo cellfree system, whereas mRNA from the cells that do not produce lambda 2 chains directed the synthesis of lambda 1 F only. DNA from the parental MOPC 315 cells and from the lambda 2 chain-producing cells contained discrete EcoRI restriction fragments coding for rearranged lambda 1 and lambda 23 chain genes and their respective germ-line V and J-C regions. DNA from the no-Ig-producing cells contained fragments coding for the rearranged lambda 1 chain gene and the germ-line V lambda 2 region, but it lacked the sequences coding for the rearranged lambda 2 chain gene and the germ-line V lambda 1 and J-C lambda 1 regions. These results suggest that rearrangements of the lambda 1 and lambda 2 chain genes occur on different chromosomes in MOPC 315 cells and imply that rearrangements of the lambda 1 and lambda 2 chain genes on the same chromosome may be mutually exclusive.     

The structure of the variable gene for the kappa chains of antibodies produced by hybridoma PTF-02

The genome of hybridoma PTF-02 has two genes for the kappa chains, and only one of these codes for the synthesis of the antibody light chains. The nucleotide sequences corresponding to the leader peptide and to the variable region of this gene were determined. An amino acid sequence corresponding to exons has been proposed on the basis of the nucleotide sequence. A nucleotide sequence adjacent to the gene at the 5'-end has also been determined, in particular, the precise localization of TATA- and CAT-boxes as well as those of the conservative deca (dc) and pentadeca (pd) sequences. The structure of the regulatory region in the gene is similar to that in the myeloma genomes. However, the 5'-region differs in its nucleotide composition and in the frequency of dc sequences from the DNA sequences adjacent to the 5'-end of eukaryotic genes which do not belong to the immunoglobulin family.     

Monoclonal antibodies against pig transierrin. Blocking and binding activity

Monoclonal mouse anti-pig transferrin antibodies PTF-01, PTF-02 and PTF-03 and anti-human transferrin antibody HTF-14 detect transferrin coupled with Sepharose particles in an indirect immunofluorescence test. Only the PTF-03 antibody can be used for immunofluorescence detection of pig transferrin bound to specific receptors on the plasma membrane. The binding of iodinated pig transferrin to PK cells was studied. It could be blocked by nonlabelled transferrin in excess, by pig serum or by anti-pig transferrin monoclonal antibodies. PTF-03 expressed the lowest blocking activity among the antibodies tested.     

Expression of a recombinant murine IgE in transfected myeloma cells

We constructed a recombinant gene encoding an immunoglobulin (Ig) heavy chain consisting of the variable region from the phosphorylcholine (PC)-specific secreting myeloma MOPC167 and the epsilon constant region from SJL mice. This gene, cloned into the shuttle vector pSV2gpt, was transfected into J558L myeloma cells, and stable transformants that expressed the epsilon gene were cloned. The IgE heavy chain in these transformants is associated with the endogenous lambda light chain and is secreted as an intact IgE molecule. However, the secreted IgE does not bind to PC conjugated to bovine serum albumin (PC-BSA). The MOPC167 kappa chain gene was cloned into the shuttle vector pSV2neo and was transfected into the epsilon heavy-chain transformant. Stable transformants were cloned that expressed both the epsilon heavy chain and the kappa light chain. IgE secreted from such a transformant was shown to bind to PC-BSA. Both types of secreted recombinant IgE bound to rat basophilic leukemia (RBL) cells, but only the IgE produced by the cell line transformed with the MOPC167 kappa gene could be cross-linked with PC-BSA to cause serotonin release.     

Secretion of a lambda 2 immunoglobulin chain is prevented by a single amino acid substitution in its variable region

We have studied two derivatives of the IgA (lambda 2) secreting myeloma cell line MOPC315:MOPC315.26, which produces and secretes a lambda 2 light chain, and MOPC315.37, which produces but does not secrete the lambda 2 chain. It has been reported that the only alteration in the MOPC315-37 lambda 2 chain is located in the variable region (Mosmann and Williamson, (1980) Cell 20, 283-292). In order to determine the nature of this alteration, we cloned the fragment of the chromosome containing the rearranged lambda 2 gene from both the nonsecreting variant MOPC315-37 and the normal lambda 2-secreting parent MOPC315-26 and determined their nucleotide sequence. We found that the nucleotide sequences coding for the leader peptide and for the constant region of the lambda 2 chain were identical in the secretor and nonsecretor. The sequences of the variable region differed at a single base pair corresponding to the first nucleotide in the codon for amino acid number 15. MOPC315-26 has a G in this position creating the codon GGT which codes for glycine, and MOPC315-37 has a C in this position creating the codon CGT which codes for arginine. Thus, we have demonstrated that a single amino acid substitution of a neutral amino acid, glycine, for a positively charged amino acid, arginine, results in the failure of a protein to be secreted.     

Gene transfer of immunoglobulin light chain restores heavy chain secretion

Several lines of evidence suggest that immunoglobulin (Ig) light (L) chain plays a role in the secretion of heavy (H) chain. For example, myeloma variant lines, which synthesize the Ig H chain but not the L chain, fail to secrete H chain protein. Here we have tested directly the role of chain assembly in the control of Ig secretion by the transfer of functional L chain genes into two such L chain-defective myeloma mutants. A lambda 2 or kappa L chain gene was introduced into variant lines of the mouse myelomas MOPC 315 (IgA, lambda 2) or PC7 (IgM, kappa), respectively. Although the two mutant lines are unable to secrete the H chain they produce, rescue of secretion of complete Ig protein molecules (IgA or IgM) was observed after transfection. These results imply that the secretory apparatus of these cells is intact and that the failure to secrete free H chain reflects a structural feature intrinsic to that protein. The implications of these results with respect to control of secretion of multi-subunit proteins are discussed.     

Density comparisons of heavy chains of membrane and secreted immunoglobulins of mouse.

In order to explore structural differences between membrane and secreted immunoglobulins the buoyant densities of mouse immunoglobulin (Ig) heavy (H) chains were compared by isopycnic centrifugation in CsCl containing guanidine hydrochloride. The buoyant densities, under denaturing conditions, of mouse myeloma protein MOPC 21 IgG, MOPC 315 IgA and MOPC 104E IgM H chains were consistent with their carbohydrate contents. Mouse membrane IgM and MOPC 104E-secreted IgM H chains were of equal density. The buoyant densities of MOPC 104E-secreted IgM and spleen-cell-secreted IgM H chains were indistinguishable. The IgD-like membrane H chain was denser than membrane IgM H chain, and its carbohydrate content was calculated to be 15.5%. The resolution of the technique was sufficient to conclude that the apparent 1500 mol.wt. difference, as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, between membrane and secreted IgM H chains was due to peptide rather than to carbohydrate. The results also imply that intact membrane IgM and IgD bind detergent and are thus integral membrane proteins.     

Cloning of a gamma 2b gene encoding anti-Pseudomonas aeruginosa H chains and its introduction into the germ line of mice

A complete, functional gamma 2b gene (pVCM) was cloned from a mouse hybridoma (VD93) with antibody activity to Pseudomonas aeruginosa. DNA sequencing of the VDJ region of pVCM determined that the VH gene was a member of the J558 family rearranged to JH2. Upon transfection into myeloma cells the gamma 2b gene gave rise to high levels of gamma 2b mRNA and gamma 2b protein. The gamma 2b protein had the same IEF pattern as the parent hybridoma protein VD93 and the antibodies formed from a combination of the pVCM gamma 2b chains and the myeloma lambda-chains bound weakly to P. aeruginosa. However, the hybrid antibodies did not discriminate between the serotypes 2 and 3, whereas the parent protein was specific for serotype 3. Transgenic mice were produced with the pVCM gamma 2b gene which expressed the gamma 2b mRNA (both membrane and secreted forms) only in lymphoid organs. However, contrary to expectations, the gamma 2b mRNA levels were higher in T cells than in B cells in three different transgenic lines. The serum of the transgenic mice had no activity to P. aeruginosa indicating the importance of L chains for the conformation of the Ag binding site. These gamma 2b transgenic mice provide a convenient tool for the study of feedback inhibition of Ig gene rearrangement.     

Cloning of immunoglobulin kappa light chain genes from mouse liver and myeloma MOPC 173

The organization of the kappa chain constant region gene was compared in DNA from an immunoglobulin-producing mouse myeloma (MOPC 173) and from liver. In situ hybridization using the Southern blotting technique revealed constant region gene-containing EcoRI-DNA fragments of 14 and 20 kb in the myeloma tissue whereas one EcoRI-DNA fragment with a length of 15 kb was found in liver DNA. After enrichment by RPC-5 chromatography and preparative electrophoresis the 14 kb fragment from MOPC 173 DNA and the 15 kb fragment from liver DNA were cloned in the bacteriophage lambda vector Charon 4A using in vitro packaging. Extensive characterization of the two fragments by restriction endonuclease mapping, in situ hybridization, and electron microscopy (R-loop and heteroduplex) showed that both fragments contain the constant region but no MOPC 173 variable region gene. Both fragments are homologous over a length of 12.5 kb including the constant region but differ from one another starting about 2.7 kb from the 5' end of the constant region gene. This indicates that the 14 kb EcoRI-DNA fragment from the myeloma tissue clearly resulted from somatic DNA rearrangement although it does not seem to carry the MOPC 173 variable region gene. These observations suggest that somatic DNA rearrangement of immunoglobulin light chain genes can involve both homologous chromosomes.Images     

Evidence for noncontiguous variable and constant region genes in both germ line and myeloma DNA.

A study has been made of the hybridization of mouse light chain cDNA to restriction enzyme digests of DNA. DNA was digested with B. st (specificity GGATCC) and fractionated on preparative agarose gels for hybridization analysis. Experiments with liver or kidney DNA yielded two peaks of hybridization with V+C cDNA corresponding to the C_κ gene and to the germ line V gene homologous to the MOPC21 V gene. Since there is no site for digestion by B. st within the MOPC21 V or C genes, this result shows that the germ line DNA carries separately the V²¹ and C_κ genes.The hybridization profiles of two different myeloma DNAs (MOPC21 and AdjPC5) differed from those of the germ line DNA. In both myelomas, only one hybridization peak was observed and no peaks corresponding to the germ line pattern were seen. The new pattern of hybridization implies that the events involved in maturation of antibody-producing cells includes rearrangement of the V and C genes.To study whether this proposed rearrangement of DNA results in contiguous V and C genes in producing cells, discrete V+C cDNA size classes (prepared with MOPC21 mRNA) were hybridized to both unfractionated restriction digested MOPC21 DNA and to the partially purified L chain gene of MOPC21 DNA. The length of the cDNA rendered resistant to single-strand-specific S₁ nuclease was determined. In no case was the full length of V+C cDNA protected from nuclease; instead, a fragment of about 290 bases (C region length) plus smaller fragments were generated. These results indicate that the rearrangement of L chain genes, which seems to occur in myeloma cells, may well not produce contiguous V and C genes in the DNA.     

Somatic point mutations in unrearranged immunoglobulin gene segments encoding the variable region of lambda light chains.

Somatic point mutations are usually found in the coding and flanking regions of functionally and aberrantly rearranged immunoglobulin variable region gene segments. Mutations in the unrearranged V gene segments of myelomas or hybridomas have not been described so far. We have cloned and sequenced unrearranged V lambda gene segments from several cell lines. There were no nucleotide changes in four unrearranged V lambda segments: one V lambda 1 from a lambda 3-producing hybridoma and one V lambda 2 from a lambda 1-producing myeloma (J558) and two V lambda 2 from a kappa-producing myeloma (P3X63). However, we found somatic mutations in the unrearranged V lambda segments from the lambda 2-producing myeloma MOPC315. The unrearranged V lambda 1 gene segment had two mutations in the coding region and the unrearranged V lambda 2 had one mutation in the 3' flanking region. We also cloned and sequenced the unrearranged J lambda and C lambda gene segments of MOPC315 and found no sequence alterations. This is consistent with the notion that the overall mutation rate is not higher in this cell line. Therefore, we suggest that the somatic hypermutation system can use unrearranged V gene segments as substrates. The extensive sequencing required for this work revealed a number of errors in the reported nucleotide sequences of the Ig lambda locus in BALB/c mice.     

Retention and loss of immunoglobulin heavy chain alleles in helper T cell hybridoma clones

Retention or loss of immunoglobulin heavy chain genes was studied in 20 functional T cell hybridoma clones. DNA probes representing C mu, C alpha and JH genes, as well as VH subgroups II and III were hybridized with restriction enzyme fragments of hybridoma DNA by the Southern filter hybridization technique. Parental alleles of the hybridoma cells were distinguished on the basis of polymorphism of the lengths of restriction enzyme fragments. All clones retained the alleles of the lymphoma parent cell BW-5147 at all four loci. Thirteen clones lost both CH and VH alleles of the immune partner cell, whereas seven retained both VH alleles, and at least C alpha of the antigen-specific partner. Hence, T cell function in these cells is compatible with the loss of most immunoglobulin heavy chain alleles. This is interpreted to indicate either gene rearrangement and deletion, or chromosome loss. Accordingly, the T cell receptor is either controlled by two split gene loci in chromosome 12, at the two respective (5' and 3') ends of the mouse heavy chain gene family, or by a gene(s) outside chromosome 12.     

Characterization of a human T cell-specific chimeric antibody (CD7) with human constant and mouse variable regions

A chimeric human-mouse anti-T lymphocyte mAb (CHT2; SDZ 214-380) has been constructed by cloning the variable region exons of both the L and H chains from the murine hybridoma RFT2 which have CD7 specificity and reactivity with a 40-kDa Ag. The variable L chain exon was joined to the human C kappa, and the variable H chain exon was joined to the human IgG1 region exon encoding the human allotype nGlm(z), nGlm(a). The gene constructs were introduced by electroporation into SP2/0, a non-Ig-producing murine myeloma. The identical tissue reactivity of the newly made CHT2 and the original murine RFT2 mAb (CD7) was confirmed by blocking experiments as well as by immunohistology and flow cytometry. Because this new mAb may have clinical use, the CD7 Ag expression of T lineage cells has also been quantitated in double and triple immunofluorescence assays in combinations with mAb to restricted forms of leukocyte common Ag that designate unprimed (CD45R+) and primed T lymphocyte populations (UCHL1+). CHT2 shows very strong reactivity with large thymic blast cells and cortical thymocytes from which T-ALL originates. Strong staining is seen on CD45R+ unprimed "virgin" T lymphocytes, whereas the expression on UCHL1+ primed "memory" cell types is weaker unless these cells are reactivated by mitogens or Ag. Thus CHT2 may spare a substantial population of resting memory T cells which is relevant to its potential therapeutic use. In addition the chimeric antibody had a greater in vitro antibody dependent cytotoxicity and a prolonged half-life (4.2 to 5.0 days) in Rhesus monkeys.     

Variable regions of antibodies to synthetic polypeptides. II. Analysis of variable region genes encoding antibodies specific for (T,G)-A--L

Monoclonal antibodies specific for the synthetic polypeptide antigen (T,G)-A--L have been produced in two strains of mice, C57BL/10 and C3H.SW. The genes encoding the variable (V) regions of these antibodies have been studied by using the DNA hybridization technique of Southern, as well as by gene cloning and sequencing. Hybridization of DNA from 14 different cell lines with a kappa-chain probe revealed that the different cell lines used one of two different gene rearrangements to encode the recombined V region gene. There was a perfect correlation between light chain rearrangement, idiotype expression, and fine specificity. Hybridization analyses of the heavy chain revealed a more complex pattern. Seven hybridomas had the rearranged heavy chain V region genes on a 4.4 kb EcoRI restriction fragment. Others were found on restriction fragments that differed in length by several hundred base pairs. The recombined heavy chain V region genes were cloned from three different hybridoma cell lines secreting anti-(T,G)-A--L antibodies, all of which express the same idiotype and fine specificity pattern. Restriction mapping and sequencing indicate that all three utilize the same V gene, identified as the 186-2 germline gene. However, different D and J genes are used to encode each of the antibodies. In contrast to the results seen in other antigen systems, heavy chain D and J genes do not have a major influence on idiotype expression and fine specificity of antibodies to the synthetic polypeptide (T,G)-A--L.     

Complete nucleotide sequence of mouse immunoglobulin mu gene and comparison with other immunoglobulin heavy chain genes

We have determined the complete nucleotides sequence (2168 bases) of the immunoglobulin mu gene cloned from newborn mouse DNA. The cloned 13kb fragment contained the entire constant region gene sequence that is interrupted by three intervening sequences at the junction of domains as previously shown in the gamma 1, gamma 2 b and alpha genes. The amino acid sequence predicted by the nucleotide sequence agrees with that of the mu chain secreted by a myeloma MOPC104E except for 8 residues out of 448 residues. The homologous domains of the mu, gamma 1 and gamma 2b genes are more similar to each other than the different domains of the mu genes are. The result implicates that the class of the immunoglobulin heavy chain genes diverged after the heavy chain genes established the multi-domain structure. The short intervening sequences of the mu and gamma genes are more conserved than the coding sequences except for the COOH-terminal domains. The results implicate that the nucleotide sequence of the intervening sequence is under selective pressure, possibly to maintain a secondary structure of the nuclear RNA to be spliced.