Reuven Laskov

Temperature-sensitive mutants of reovirus type 3: Studies on the synthesis of viral peptides

Abstract The synthesis of the major reovirus peptides has been examined in cells infected with ts mutants containing lesions in five cistrons. Immunological precipitation has been used so that peptides made in small amounts could be detected. At the permissive temperature, the mutants did not significantly differ from the wild type. At nonpermissive temperatures, different mutants produced different amounts of viral peptides, but all of the major peptides were made in the mutants examined and the size of each of these peptides was the same as in the wild type.

Immunoglobulin Production: Method for Quantitatively Detecting Variant Myeloma Cells

Mouse myeloma cells in continuous culture were cloned in soft agar. The clones were assayed for their ability to synthesize heavy and light chains of gamma globulin by immunoprecipitation directly in the agar. A minor population secreting only light chains was identified. The two cell types were recloned and a low incidence of conversion of 7S to light-chain production was demonstrated. This technique can be used to isolate rare variants of cells secreting specific macromolecules.

Extinction of Ig genes expression in myeloma x fibroblast somatic cell hybrids is accompanied by repression of the oct-2 gene encoding a B-cell specific transcription factor.

In most instances, fusion of differentiated cell types with fibroblasts has resulted in the extinction of differentiation‐specific traits of the nonfibroblast parental cell. To explore the genetic basis of this phenomenon, we have used a series of somatic cell hybrids between myeloma cells and fibroblasts. Previous findings show that in these hybrids expression of the immunoglobulin (Ig) genes was extinguished at the transcriptional level. Our present results show that NF‐kappa B transcription factor, known to be critical for kappa‐chain enhancer activity, is present although in a lower amount, in the nucleus and in the cytosolic fraction of most of these hybrids (probably attached to the previously postulated I‐kappa B inhibitor). In contrast, the expression of the NF‐A2/OTF‐2 transcription factor encoded by the oct‐2 gene, which binds to the octameric motif located in the Ig promoters and heavy chain gene enhancer, is extinguished at the transcriptional level. Our data thus suggest that extinction of Ig genes expression occurs via an indirect mechanism in which a fibroblast factor suppresses transcription factor(s) which are critical for Ig transcription.

Synthesis, assembly and secretion of gamma globulin by mouse myeloma cells. II. Assembly of IgG2b immunoglobulin by MPC 11 tumor and culture cells.

Abstract The kinetics, pathway, and efficiency of covalent assembly of mouse IgG 2b immunoglobulin has been investigated using both tumor and cultured cells from the MPC 11 mouse myeloma. More than half of the interchain disulfide bonds were formed within three to five minutes after the synthesis and release of newly synthesized heavy and light chains and assembly was largely completed within 10 to 20 minutes after synthesis. Multiple pathways of assembly were used. Cells derived from the tumor are blocked in the covalent assembly of half molecules into IgG, but carry out the non-covalent assembly of half molecules to a higher polymer. This block in assembly may be due to the presence of a large excess of light chains rather than to a structural defect in the polypeptide chains.

Extinction of expression of immunoglobulin genes in myeloma X fibroblast somatic cell hybrids.

Adherent hybrids between immunoglobulin-producing mouse myeloma cells and fibroblasts do not produce immunoglobulin polypeptide chains. These hybrids retained the actively rearranged immunoglobulin genes of the myeloma parental cells but lacked immunoglobulin heavy- and light-chain RNA transcripts. We conclude that the shutoff of immunoglobulin production in these hybrids occurs at the transcription or early processing level.

Production of Monoclonal Antibodies Against Enamelin and Against Amelogenin Proteins of Developing Enamel Matrix

The extracellular matrix of developing enamel contains two major classes of proteins, the hydrophobic proline-rich amelogenins and the acidic serine-, glycine-, and aspartic-rich enamelins. These proteins have been postulated as playing a major role in the mineralization and structural organization of developing enamel. To identify and further characterize these different proteins and their possible role in this complex process of biological mineralization, we have in recent years been concerned with the production of specific probes for these proteins. Previously, we have reported on the successful production of specific polyclonal antibodies against enamelin proteins, which did not cross-react with amelogenins, and against amelogenin proteins, which did not cross-react with enamelins (Deutsch et al., 1986, 1987). We now report the production of monoclonal antibodies against a major bovine amelogenin protein (28 kDa) and against a major bovine enamelin protein (66 kDa). One monoclonal antibody against amelogenin and one against enamelin are described. The results showed that the monoclonal antibody against the amelogenin protein reacted strongly with the 28-kDa amelogenin protein band but did not cross-react with enamelins, and the one against the enamelin protein reacted with the 66-kDa enamelin protein but did not cross-react with amelogenins. These monoclonal antibodies provide a specific and powerful tool to distinguish between and further characterize these different classes of proteins, and to improve our understanding of the process of enamel formation.

The regulation of immunoglobulin synthesis and assembly.

Individual mouse and human myelomas provide a series of “variants” which, when compared with one another, can lead to a better understanding of the mechanisms involved in the synthesis, assembly and secretion of the immunoglobulin molecule.17 2 We have therefore compared twelve IgG producing mouse tumors and two cultured cell lines with respect to: (1) the relative amounts of immunoglobulin and cell protein produced; (2) the relative number of heavy (H) and light (L ) chains synthesized; (3) the identity of the partially assembled immunoglobulin molecules found within the cell and secreted into the medium; and (4) the pathways and kinetics of assembly of IgG.3 Although each tumor differed from the others in one or more of these parameters, the average behavior of all the tumors resembled closely that of lymph node cells obtained from normal immunized mice. A second conclusion drawn from these studies was that the major precursor of the IgG,, subclass of immunoglobulin was half molecules (HL) , while the predominant precursor of the IgG, and IgG,, subclasses was H chain dimers (H,) . However fruitful a comparison of the different mouse tumors and cell lines proved to be, considerably more information would be forthcoming if genetic tools could be developed similar to those used to study microorganisms. To this end, we have adapted the MPC-11 mouse myeloma tumor to continuous culture,‘ cloned the cultured cells in soft agar, and developed a plate detection method to identify and quantitate rare variants of immunoglobulin product i ~ n . ~ ? ti These studies revealed that there was a stepwise loss of the ability to produce H and L chains and the rate of “mutation” from H + L chain production to L chain production was 1.1 X 10-3/cell/generation.8

Extinction of B-cell surface differentiation markers in hybrids between murine B-lymphoma and myeloma cells

Abstract Fusion between murine B-lymphoma cells bearing membrane IgM with either IgG or light chain secreting myeloma, resulted in cell hybrids synthesizing and secreting large quantities of IgM. In contrast, the hybrids did not secrete IgD even though it is also present on the surface of the B-lymphoma cells. B-Cell surface markers such as the IgM, IgD, Ia and the Fc receptor, which were present on the B-lymphoma cells, but not the myeloma cells were not expressed on the surface of the hybrids. Hybrids which secrete IgM and retain the B-cell membrane differentiation antigens were not detected, even when selection was done under conditions which favor the growth of the lymphoma parental cells.

Production of anti-RNA antibody by hybridoma cells: Purification from mixed immunoglobulin products

Abstract Fusion between spleen cells from an autoimmune NZB/NZW mouse and the Balb/c drugresistant MPC-11 myeloma resulted in the formation of a hybridoma-secreting RNA-specific IgG-3 antibody and the parental IgG-2b myeloma. Analysis of the mixed immunoglobulin assembly products made by the hybridoma cells showed efficient pairing of IgG-2b and IgG-3 heavy chains and did not show a marked preferential assembly of the homologous heavy and light chains. Partial purification of the anti-RNA antibody from the mixed assembly products was achieved by utilizing an antigen affinity column (RNA-Sepharose). The use of a heavy chain-specific affinity column (anti-IgG-2b-Sepharose) increased the purity of the desired antibody, but parental light chains were still present after this step. A complete purification of the RNA-binding protein could be achieved by papain cleavage of the total IgG fraction and binding of the resulting Fab fragments to RNA-Sepharose. This procedure may, therefore, be employed as a general method for purifying antibodies from hybridomas that continue to produce their parental myeloma chains.

Characteristic surface morphology of human and murine myeloma cells: a scanning and transmission electron microscopic study.

Summary. Cells from cultured human and murine myeloma cell lines and circulating leukacmic plasma cells from four patients with generalized myeloma were studied by transmission and scanning electron microscopy and time‐lapse cinematography. Both circulating and cultured cells exhibited consistent surface architectures and varying numbers of prominent blebs of different sizes were seen, in addition to microvilli. The presence of surface blebs appears to be a characteristic feature for secreting and non‐secreting myeloma cells.