Immunologic memory to PC-KLH: participation of the Q52 VH gene family.

BALB/c mice immunized with phosphocholine-conjugated keyhole limpet hemocyanin respond with two major groups of antibodies that differ with respect to fine specificity and idiotype. Group I antibodies predominantly bear the T15 idiotype, and show appreciable affinity for the haptens PC and nitrophenyl PC (NPPC), whereas group II antibodies have appreciable affinity for NPPC only and are T15 idiotype negative. Previous studies indicated that group II binding characteristics may derive from the use of novel V gene segments not observed in group I antibodies. To determine the nature of VH gene usage in the group II antibody response, we examined the VH region of a prototype group II hybridoma, PCG1-1. The nucleotide sequence obtained from the VDJ region indicates that PCG1-1 utilizes a VH gene not observed in the group I response, one that belongs to the Q52 VH family. The PCG1-1 VH nucleotide sequence shares 97% identity with the myeloma M141 VH gene. In addition, PCG1-1 utilizes a D segment most closely related to DSP2.6 rearranged to JH-3. These data indicate that M141, a VH gene not seen in group I anti-PC antibodies is utilized by PCG1-1 to generate a PC-protein-binding group II antibody. PCG1-1 was previously shown to express the V kappa 1-3 light chain, a characteristic shared by several group II hybridomas. Furthermore, here we examined the VH gene rearrangements in four lambda 1-bearing group II hybridomas that share a common JH rearrangement with PCG1-1 by Southern blot analysis. A VH-specific probe that detects M141 VH rearrangements revealed that all four lambda 1 hybridomas as well as PCG1-1 share an identical VH gene rearrangement to JH-3. Thus the M141 VH gene product is able to utilize two distinct light chains to generate group II-like combining sites.

A cloned cDNA probe for rat immunoglobulin epsilon heavy chain: construction, identification, and DNA sequence.

IgE has clinical importance because it is responsible for immediate hypersensitivity. Studies of IgE expression in rats appear to contradict current models for immunoglobulin gene expression. To study rat IgE expression at the RNA and DNA levels, we have constructed a cDNA for part of the rat epsilon (epsilon) heavy chain that is expressed by a rat myeloma, IR162. The rat epsilon-chain clone was initially identified by an efficient selection scheme. DNA sequencing of the 580-bp cDNA revealed that it encoded 176 amino acids that were 50% homologous to the human epsilon H chain. The sequence begins near the end of the CH2 domain and ends 31 amino acids into the CH4 domain. Cysteines important for the structure of the human IgE were conserved in the rat epsilon H-chain. The identity of the cloned epsilon cDNA was confirmed by comparison with a portion of the constant region gene for mouse epsilon H chain. The mouse and rat nucleotide sequences were 79% homologous.

Physical and biological properties of an epsilon-heavy chain mRNA and a kappa-light chain mRNA coding for rat immunoglobulin E.

The mRNA coding for epsilon-heavy chain and kappa-light chain have been highly enriched from a rat IgE-producing myeloma, IR-162. Based on denaturing gel analyses, the 20 S epsilon-heavy chain mRNA has an estimated molecular weight of 850,000, equivalent to about 2500 nucleotides. The 14S rat kappa-light chain mRNA has an estimated molecular weight of 410,000, equivalent to about 1200 nucleotides. Only about two-thirds of the length of these mature cytoplasmic rat mRNA code for protein. The 20 S mRNA stimulates the in vitro synthesis of a single major serologically related protein which is large enough to be epsilon-heavy chain. It is unglycosylated and has an apparent molecular weight of about 62,000. The in vivo unglycosylated epsilon-heavy chain, obtained in the presence of tunicamycin, has an apparent molecular weight of about 59,000, compared with about 76,000 for the glycosylated heavy chain of the secreted rat IgE. Therefore, the in vitro synthesized epsilon-heavy chain protein is about Mr = 3000 larger than the in vivo unglycosylated epsilon-heavy chain, equivalent to about 25 extra amino acids. This is consistent with the synthesis of an epsilon-heavy chain putative precursor. Likewise, the 14 S mRNA stimulates the in vitro synthesis of a single putative precursor protein, which is serologically related to kappa chain, is unglycosylated, and is about an extra 20 amino acids. This is the first report on the physical and biological properties of an epsilon-heavy chain mRNA, as well as any rat immunoglobulin mRNA.

Mouse immunoglobulin mu heavy chain mRNA of Y5781, a high yield myeloma.

The BALB/c myeloma tumor, Y5781, has a high level of mu heavy chain mRNA and kappa light chain mRNA, as suggested by denaturing gel analyses of poly(A)-rich, total polysomal mRNA, and confirmed for the mu heavy chain mRNA by kinetic complexity analyses. Both the mRNA coding for the heavy and light chains appear as very prominent and discrete peaks above the generally polydisperse background of the total polysomal mRNA. This mRNA level appears to be stable through a limited number of subcutaneous passages of this myeloma, providing a potentially useful system for mu heavy chain mRNA synthesis and processing. The mu heavy chain mRNA of this myeloma has been enriched to about 60% homogeneity by physicochemical means. In agreement with a previous report (Faust, C.H., Jr., Heim, I., and Moore, J. (1979) Biochemistry 18, 1106-1119), the following physical and biological properties were observed. The mature cytoplasmic mu heavy chain mRNA is 950,000 daltons, i.e. about 2800 nucleotides, and contains approximately 800 undefined, nontranslated bases. In an mRNA-dependent cell-free system, this mRNA stimulates the synthesis of a single, serologically reactive mu heavy chain-like protein, confirmed by tryptic peptide maps.

No detectable reiteration of genes coding for mouse MOPC 41 immunoglobulin light-chain mRNA.

RNA fractions rich in immunoglobulin light (L)-chain mRNA were isolated from mouse myeloma MOPC 41 by procedures previously described, and chemically labeled with 125I. These RNA fractions were hybridized with MOPC 41 DNA under conditions of DNA excess. Hybridization conditions were chosen under which the entire sequence of the L-chain mRNA probe, thus including the variable region, remains available for hybridization throughout the reaction. The hybridization (C0t) curve showed double transition kinetics, with one component corresponding to about 250 gene copies and the other to about two to four copies. In contrast, when MOPC 41 L-chain mRNA was further purified as a single band by gel elecptrophoresis in 99% formamide, the hybridization curve showed only a single transition, corresponding to about two to four genes, with the disappearance of the "reiterated" component. That component resulted therefore from contaminating RNA species. The data indicate that no reiteration can be detected by RNase or by hydroxylapatite for the genes corresponding to the entire sequence of MOPC 41 L-chain mRNA, including the untranslated segments, within the limits of detectability of short reiterated segments. It thus appears that there is only one or very few genes corresponding to the 41 L-chain variable region "subgroup" in MOPC 41 DNA. The possibility that the variable genes of plasmocytes might result frm a combination of several nonreiterated germline genes is discussed.

Estimation of the number of genes coding for the constant part of the mouse immunoglobulin kappa light chain.

As previously shown, purified 14S RNA of mouse myeloma MOPC-41 forms a single peak on sucrose gradients and gel electrophoresis and codes for a single polypeptide chain, the immunoglobulin light chain produced by the same myeloma in vivo. This 14S mRNA was used for the enzymatic synthesis of DNA (cDNA) which is complementary to the RNA template. A DNA fraction was isolated which has an average size of 300 nucleotides. Kinetic studies of the hybridization of the 14S RNA with the cDNA indicate that about 40% of the RNA consists of a single RNA sequence. From the size of the cDNA fraction and from the direction of DNA synthesis, it can be concluded that the cDNA includes the sequence complementary to the constant region of light chain mRNA. This radioactive cDNA was used for DNA.DNA reannealing experiments with unlabeled DNA from mouse liver or myeloma tumor in 3 x 10(6)-fold excess. This allowed the determination of the number of copies in the mouse genome of those sequences represented in the cDNA. The data show no significant reiteration in either liver or myeloma DNA and suggest that the gene coding for the constant part of immunoglobins is present in the haploid genome in one to five copies. Furthermore, this gene is not "amplified" in nuclear DNA of myeloma plasmocytes.

Enzymatic synthesis of DNA complementary to purified 14S messenger RNA of immunoglobulin light chain.

The 14S messenger RNA, which contains poly(adenylic acid), of MOPC 41 (mouse plasmocytoma) immunoglobulin light chain, purified to a single peak as shown by polyacrylamide gel electrophoresis, was used to synthesize complementary DNA with the RNA-dependent DNA polymerase of avian myeloblastosis virus. DNA synthesis is entirely dependent on added RNA template and oligo(dT) primer. Both the size and the concentration of the primer affect the reaction. The product behaves similarly to DNA during centrifugation in cesium sulfate density gradients. It is shown by hybridization that the DNA made is complementary to the purified template, light-chain mRNA. The high specific activity of the complementary DNA should make it suitable for gene-dosage experiments. According to alkaline sucrose gradient analyses, some complete complementary DNA transcripts of the 14S mRNA seem to be made. Oligo(dG) can also function as a primer for DNA synthesis, possibly by annealing to an internal cluster of cytidines in the mRNA, that correspond to the bases coding for amino-acids 119 and 120 of the MOPC 41 light chain.