Scope of action of the immunoglobulin mutator system.

The authors have developed a method to measure the rate of spontaneous mutations taking place in IgH, the gene encoding the immunoglobulin heavy chain. When an amber chain-termination codon mutates to a sense codon, translation of the polypeptide chain will be completed, and mutant cells producing the heavy chain can be detected with a fluorescent labelled antibody. The protocol used is the compartmentalization test which minimizes any effect of selection. In subclones of the pre-B lymphocyte line 18-81, the spontaneous mutation rate in the part of IgH encoding the variable region is somewhat greater than 10(-5) mutations per base pair per generation. This supports the hypothesis that hypermutation is not dependent on cell stimulation by an antigen. In a hybrid between a cell of this line and a myeloma (which represents the terminal stage of the B-cell lineage), the mutation rate was too low to be determined by this test, less than 10(-9). When the same loss to gain procedure system was used with an opal chain-terminating codon in the part of IgH encoding the constant region (C mu), a high rate of reversion by deletion was found. Long (more than one exon) and short (less than one exon) deletions occurred at rates of 1.7 x 10(-5) and 1.4 x 10(-7) per generation, respectively. It is thought that the high rate of deletion is not related to somatic hypermutation but rather to DNA rearrangement during the heavy-chain class switch, which is occurring in these pre-B cell lines. The point mutation rate was too low to be detected above the background of deletion mutants, less than 5 x 10(-8). The immunoglobulin mutator system works weakly, if at all, on two other, nonimmunoglobulin, genes tested: B2m (beta 2 microglobulin) and the gene for ouabain resistance.

V lambda 2 rearranges with all functional J lambda segments in the mouse.

We have analyzed 210 lambda-producing hybridomas derived from lipopolysaccharide-stimulated spleen cells from a single kappa-suppressed mouse. All were classified as lambda 1, lambda 2 or lambda 3 with the exception of four unusual lines. Two of these were due to V lambda 2 J lambda 1 and the other two to V lambda 2 J lambda 3 rearrangements. The lines were clonally independent since the point of VJ recombination in each one was different. Southern blot analysis of the V lambda 2 C lambda 1-producing lines showed no evidence for an inversion. Under the assumption of a simple deletion model of rearrangement these findings place the V lambda 2 cluster upstream of the V lambda 1 cluster oriented in the same direction.

Immunoglobulin heavy chain toxicity in plasma cells is neutralized by fusion to pre-B cells.

A plasma cell hybridoma frequently loses its immunoglobulin heavy (H) chain spontaneously but rarely is production of its light (L) chain lost. Upon fusion to a pre-B-cell hybridoma that produces no Ig chain, the L chain is frequently lost. In cells without the L chain the H chain, which is derived from the plasma cell, is not chemically modified. Our results indicate that, in pre-B cells, but not in plasma cells, there must be a mechanism that neutralizes the toxic effect of free H chain.

A Mott cell hybridoma.

A hybridoma is described that exhibits all the characteristic features of Mott cells. It has spherules (Russell bodies) in the cytoplasm made up of dilated rough endoplasmatic reticulum and containing condensed immunoglobulin (lambda 1 light chains). Some of the cells appear to be very fragile, and free spherules are often found on cell smears. Cells with the Mott cell characteristics are still able to divide, but they do not secrete immunoglobulin. Hybridomas of this kind should be useful for determining the place of the Mott cell within the scheme of B cell differentiation.

Expression of immunoglobulin heavy chain at a high level in the absence of a proposed immunoglobulin enhancer element in cis.

The major intron between the J and C gene segments of the immunoglobulin heavy (H) chain locus contains an enhancer-like sequence, and it has been proposed that this enhancer is necessary to achieve high levels of H chain expression. We have isolated a subclone of the lymphoid pre-B cell line 18-81 that lacks this enhancer but nevertheless produces mu chain at the level characteristic of pre-B cells. Another subclone with a larger deletion does not produce mu chain, but upon fusion with a myeloma that does not produce any immunoglobulin chain, mu chain is expressed by the homolog from the pre-B subclone. The hybridoma lacks the proposed enhancer element in cis; nevertheless it produces as much mu chain as other plasma cell hybridomas. Therefore, this enhancer element is not obligatory for a high level of H chain production.

Allelic inclusion in the pre-B-cell line 18-81.

In an Abelson-virus-transformed mouse lymphoid cell line with pre-B-cell characteristics, a few cells continuously produce heavy chains from both homologs. Each chain has a different variable region. These cells thereby exhibit allelic inclusion rather than allelic exclusion.

Immunoglobulin heavy-chain class switching in a pre-B cell line is accompanied by DNA rearrangement.

Switching of an Abelson virus-transformed mouse lymphoid cell line from immunoglobulin mu to gamma 2b heavy-chain synthesis in vitro is accompanied by loss of DNA sequences between the JH and C gamma 2b gene segments, and thus cannot be explained by differential RNA processing. The light-chain loci of both mu- and gamma 2b-producing cell clones are in the embryonic configuration, which indicates that class switching can occur in pre-B cells.

Allelic exclusion of immunoglobulin expression is not caused by somatic segregation.

We have investigated the karyotype of immunoglobulin-producing cells in heterozygous animals. Using a karyotypic marker for one homolog of a chromosome carrying immunoglobulin genes, we established that immunoglobulin-producing cells are heterozygous with respect to this chromosome. Therefore, allelic exclusion of immunoglobulin expression cannot be caused by somatic chromosome segregation.

Mouse mitochondrial superoxide dismutase locus is on chromosome 17.

The hamster X mouse hybridoma cell line GCL28 carries only one copy of mouse chromosome 17 but expresses H-2 antigens controlled by the major histocompatibility complex of the mouse. The cell line and clones derived from it were subjected to treatment with H-2 specific antisera and complement and a series of H-2 antigen-negative clones was produced. Typing of the clones for the mouse enzyme glyoxalase 1, which is encoded by an H-2-linked gene, revealed that the loss of H-2 antigen expression was accompanied by the loss of chromosomes 17 in these clones. This suggestion was verified by karyotype analysis of selected clones. Typing of the clones and subclones for the mouse mitochondrial superoxide dismutase (SOD-2) indicated complete concordance between loss of chromosome 17 and loss of SOD-2 activity. This finding suggests that the locus controlling the expression of SOD-2 is located on chromosome 17. Since a similar locus in the human is linked to HLA, the human major histocompatibility complex, extensive homology must exist between the mouse and human MHC-bearing chromosomes.

Expression of mu and gamma immunoglobulin heavy chains in different cells of a cloned mouse lymphoid line.

A cloned cell line derived from mouse bone marrow and transformed by Abelson virus is shown to synthesize two different heavy chains, mu and gamma 2B, in vitro. This characteristic is stable because it persists upon subcloning. Although most of the immunoglobulin-synthesizing cells produce either mu or gamma 2B heavy chains, a few cells contain both heavy chains, suggesting immunoglobulin class switching. Karyotypes show a complement of 41 chromosomes. Two copies of chromosome 12, to which immunoglobulin heavy chain structural genes have been assigned, were found. No light chain was found in either the mu- or the gamma 2B-producing cells. However, fusion of the cell line with a myeloma that synthesizes neither heavy nor light chains caused expression of kappa light chain in the hybridoma synthesizing mu chain. No light chain could be detected in the hybridomas synthesizing gamma 2B heavy chain.

Simultaneous expression of mouse immunoglobulins M and D is determined by the same homolog of chromosome 12.

A hamster-mouse hybrid cell line expressiong both murine IgM and murine IgD on the membrane was shown to have only one copy of mouse chromosome 12. This chromosome is known to carry the structural genes for the immunoglobulin heavy chains. Cloning of populations selected for loss of mouse membrane IgM yielded cells that had also lost expression of membrane IgD, but not the expression of hamster immunoglobulin heavy chain. Karyotype analysis of these subclones demonstrated the concurrent loss of the chromosome 12 present in the parental hybrid. Absence of this chromosome was confirmed by use of the isozyme acid phosphatase 1. The results of the genetic analysis prove that the coexpression of mu and delta immunoglobulin heavy chains is not due to long-lived immunoglobulin mRNA nor to the transcription of genes on homologous chromosomes. We conclude that the genetic information for IgM and IgD expressed by a single cell lies on the same chromosome.

Antibody diversity in amphibians: inheritance of isoelectric focusing antibody patterns in isogenic frogs.

Anti-sheep red cell, anti-dinitrophenyl, anti-phosphorylcholine antibody responses have been followed in isogenic frogs of the genus Xenopus. Isoelectric focusing antibody patterns show a high degree of overlap for all antigens studied, and a heterogeneity that is lower than in mammals for the same antigens. Inheritance of antibody isoelectric focusing spectrotypes was demonstrated for sheep red cells and dinitrophenyl in two clones of isogenic animals. Outbred frogs show a higher frequency of spectrotype sharing than outbred mammals. It is therefore suggested that antibody diversity is lower in frogs than in mammals.

Switch in immunoglobulin class production observed in single clones of committed lymphocytes.

Mouse spleen cells, after stimulation with lipopolysaccharide, were cloned in culture. After 4 to 5 days, the daughter cells were stained and examined for immunoglobulin class with double immunofluorescent reagents. A switch of the stained color of these cells was observed, implying a switch from imunoglobulin M to immunoglobulin G production in the progeny of a single B cell.

Transplantation of nuclei from lymphocytes of adult frogs into enucleated eggs: special focus on technical parameters.

The technique of transplantation of nuclei from adult lymphocytes into enucleated eggs from Xenopus laevis (South African clawed toad) is described. The splenic lymphocytes from the one-nucleolus mutant were bound via their immunoglobulin receptors to nylon fibers, derivatized with the antigen used for immunization. A technique for coupling other cell types with Woodward reagent is also described. The cells were broken by aspiration into a micropipette and injected into enucleated eggs. The egg pronucleus was eliminated by UV treatment followed by surgical removal. The origin of the genome of developing embryos was determined on karyotype preparations by looking for the nucleolar organizer on the chromosome pair No. 12. Participation of egg pronucleus in development was frequent as judged by the incidence of gynogenetic diploid individuals and of tetraploid animals exhibiting characteristics of both recipient egg and somatic cell donor karyotypes. Apparently normal, lymphocyte-dervived tadpoles were obtained, but they died at 12 days. This shows that differentiated cells from adults, such as lymphocytes, can re-express the genes necessary for ontogeny.

Lymphocytes of the toad Xenopus laevis have the gene set for promoting tadpole development.

Nuclear transplantation experiments show that differentiated cells, such as lymphocytes, from the adult frog can express the genes necessary for tadpole development. The transplanted cells were proven to be lymphocytes by immunological methods. The origin of the tadpoles that developed after lymphocyte nuclei injections was ascertained by a karyotypic marker.

Isolation, characterization, and action of colicin M.

Colicin M was isolated from Escherichia coli K-12 32T 19F/T1. The purified, biologically active protein had a molecular weight of 27,000. It contained phosphatidyl ethanolamine. The molecular weight found for the polypeptide chain by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate was 18,000. Colicin M was found to be firmly integrated in the membrane of the producing strain. The action of the colicin seems to be on the membrane, since cells of the susceptible strain E. coli K-12 ROW/V/22.1 lyse rapidly. Using the phase contrast microscope, lysis was followed by decrease in turbidity of the cell culture and release of protein into the medium. Lysis started at about 15 min after addition of colicin M and was completed after 40 to 60 min. At this time, one-third of the protein had been released from the cells. The number of viable cells dropped within 10 min to 0.01%. Colicin M induced formation of spheroplasts in the presence of 16% sucrose. The electron microscope examination revealed that at first bulges in the cell envelope appear, most frequently occurring equatorially but also occurring at sites all over the cell. In the process of spheroplast formation, the cytoplasmic membrane often retreats from one-half of the outer membrane so that the cytoplasm is confined to one hemisphere. Sucrose did not prevent cells from dying unless cells were pregrown in a sucrose containing medium for several generations before colicin M was added. With cells pregrown in the presence of sucrose, the number of survivors was 100 times higher than in the absence of sucrose.