Biochemical genetics of the mouse IgM system

1984 ◽  
Vol 62 (4) ◽  
pp. 217-224 ◽  
Author(s):  
Marc J. Shulman ◽  
Robert G. Hawley ◽  
Atsuo Ochi ◽  
W. O. T. Baczynsky ◽  
Catherine Collins ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Heavy Chain ◽  
Mutant Cell ◽  
Immunoglobulin M ◽  
Structural Basis ◽  
Deletion Mutants ◽  
Transfer System ◽  
Biochemical Genetics ◽  
Chain Synthesis

Using a mouse hybridoma system, we have developed methods of isolating a variety of mutant cell lines in which immunoglobulin function or synthesis is defective. The analysis of mutants defective in κ chain synthesis has defined a class of murine transposons. The deletion mutants produce immunoglobulin M (IgM) bearing μ heavy chain fragments and provide information on the requirements of IgM assembly and μ gene expression. We also describe a transfer system for the μ and κ genes which will be useful in analyzing the structural basis of IgM function.

Mutations affecting the structure and function of immunoglobulin M

1982 ◽  
Vol 2 (9) ◽  
pp. 1033-1043
Author(s):  
M J Shulman ◽  
C Heusser ◽  
C Filkin ◽  
G Köhler
Keyword(s):  
Mutant Cell ◽  
Immunoglobulin M ◽  
Structural Basis ◽  
Light Chains ◽  
Partial Deletion ◽  
Structural Alterations ◽  
Specific Immunoglobulin ◽  
Synthesis And Processing ◽  
And Function ◽  
Abnormal Glycosylation

Using a hybridoma cell line which secretes hapten-specific immunoglobulin M (IgM), we have isolated a variety of mutants which produce abnormal immunoglobulin. Immunoglobulin was tested for the size and composition of the component heavy and light chains and for variable and constant region related functional and serological activities. Some mutants secrete IgM which seems to be defective in hapten binding; others make IgM which appears not to activate complement. Many of the mutants secrete monomeric as opposed to pentameric IgM. In some cases, the defect apparently correlates with structural alterations in the mu heavy chain: partial deletion, polypeptide addition, and abnormal glycosylation have been observed. These mutant cell lines provide a means of identifying the structural basis of IgM function and of studying the biochemistry of IgM synthesis and processing.

scholarly journals Mutations affecting the structure and function of immunoglobulin M.

1982 ◽  
Vol 2 (9) ◽  
pp. 1033-1043 ◽  
Author(s):  
M J Shulman ◽  
C Heusser ◽  
C Filkin ◽  
G Köhler
Keyword(s):  
Mutant Cell ◽  
Immunoglobulin M ◽  
Structural Basis ◽  
Light Chains ◽  
Partial Deletion ◽  
Structural Alterations ◽  
Specific Immunoglobulin ◽  
Synthesis And Processing ◽  
And Function ◽  
Abnormal Glycosylation

Using a hybridoma cell line which secretes hapten-specific immunoglobulin M (IgM), we have isolated a variety of mutants which produce abnormal immunoglobulin. Immunoglobulin was tested for the size and composition of the component heavy and light chains and for variable and constant region related functional and serological activities. Some mutants secrete IgM which seems to be defective in hapten binding; others make IgM which appears not to activate complement. Many of the mutants secrete monomeric as opposed to pentameric IgM. In some cases, the defect apparently correlates with structural alterations in the mu heavy chain: partial deletion, polypeptide addition, and abnormal glycosylation have been observed. These mutant cell lines provide a means of identifying the structural basis of IgM function and of studying the biochemistry of IgM synthesis and processing.

scholarly journals Comparison of cell lines deficient in antigen presentation reveals a functional role for TAP-1 alone in antigen processing.

1994 ◽  
Vol 180 (4) ◽  
pp. 1415-1425 ◽  
Author(s):  
R Gabathuler ◽  
G Reid ◽  
G Kolaitis ◽  
J Driscoll ◽  
W A Jefferies
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Heavy Chain ◽  
Antigen Processing ◽  
Mutant Cell ◽  
Antigenic Peptides ◽  
Ifn Gamma ◽  
Chain Molecules ◽  
Lung Carcinoma Cell Line ◽  
Infected Cells

Cytotoxic T lymphocytes (CTL) recognize antigenic peptides bound to major histocompatibility complex class I antigens on the cell surface of virus-infected cells. It is believed that the majority of peptides originate from cytoplasmic degradation of proteins assumed to be mediated by the "20S" proteasome. Cytosolic peptides are then translocated, presumably by transporters associated with antigen processing (TAP-1 and -2), into the lumen of the endoplasmic reticulum (ER) where binding and formation of the ternary complex between heavy chain, beta2-microglobulin (beta 2m) and peptide occurs. In this study, we have analyzed and compared the phenotype of two mutant cell lines, the thymoma cell line RMA-S and a small lung carcinoma cell line CMT.64, in order to address the mechanism that underlies the antigen processing deficiency of CMT.64 cells. Unlike RMA-S cells, vesicular stomatitis virus (VSV)-infected CMT.64 cells are not recognized by specific CTL. Interferon gamma (IFN-gamma) treatment of CMT.64 cells restores the ability of these cells to process and present VSV in the context of Kb. We show that although CMT.64 cells express a low level of beta 2m, the recognition of VSV-specific CTL is not restored by increasing the amount of beta 2m synthesized in CMT.64 cells. In addition, we find that CMT.64 cells express moderate levels of Kb heavy chain molecules, but most of it is unstable and rapidly degraded in the absence of IFN-gamma treatment. We infer that the antigen processing deficiency does not lie at the level of beta 2m or Kb production. We find also that the mRNAs for both TAP-1 and -2 are present in RMA and RMA-S cells but are absent in uninduced CMT.64 cells. Upon IFN-gamma induction, both mRNAs are highly expressed in CMT-64 cells. In addition, we find that the low molecular mass polypeptides 2 and 7, and additional components of the proteasome are induced by IFN-gamma in CMT-64 cells. Finally, introduction of the rat TAP-1 gene in CMT.64 cells restores CTL recognition of VSV-infected cells. These results indicate that a TAP-1 homodimer may translocate peptides in the ER and explain partially the CMT.64 defect and the RMA-S phenotype. These findings link a dysfunction in the transport and/or generation of antigenic peptides to the capacity of tumor cells to evade immunosurveillance and provide a unique model system to dissect this phenomenon.

scholarly journals Posttranslational association of immunoglobulin heavy chain binding protein with nascent heavy chains in nonsecreting and secreting hybridomas.

1986 ◽  
Vol 102 (5) ◽  
pp. 1558-1566 ◽  
Author(s):  
D G Bole ◽  
L M Hendershot ◽  
J F Kearney
Keyword(s):  
Monoclonal Antibody ◽  
Endoplasmic Reticulum ◽  
Cell Lines ◽  
Heavy Chain ◽  
Light Chain ◽  
B Lymphocyte ◽  
Binding Protein ◽  
Heavy Chains ◽  
Chain Synthesis ◽  
Ig Heavy Chain

A rat monoclonal antibody specific for immunoglobulin (Ig) heavy chain binding protein (BiP) has allowed the examination of the association of BiP with assembling Ig precursors in mouse B lymphocyte-derived cell lines. The anti-BiP monoclonal antibody immunoprecipitates BiP along with noncovalently associated Ig heavy chains. BiP is a component of the endoplasmic reticulum and binds free intracellular heavy chains in nonsecreting pre-B (mu+, L-) cell lines or incompletely assembled Ig precursors in (H+, L+) secreting hybridomas and myelomas. In the absence of light chain synthesis, heavy chains remain associated with BiP and are not secreted. The association of BiP with assembling Ig molecules in secreting hybridomas is transient and is restricted to the incompletely assembled molecules which are found in the endoplasmic reticulum. BiP loses affinity and disassociates with Ig molecules when polymerization with light chain is complete. We propose that the association of BiP with Ig heavy chain precursors is a novel posttranslational processing event occurring in the endoplasmic reticulum. The Ig heavy chains associated with BiP are not efficiently transported from the endoplasmic reticulum to the Golgi apparatus. Therefore, BiP may prevent the premature escape and eventual secretion of incompletely assembled Ig molecules.

scholarly journals Immunoglobulin M biosynthesis. Production of intermediates and excess of light chain in mouse myeloma MOPC 104E

1971 ◽  
Vol 123 (4) ◽  
pp. 635-641 ◽  
Author(s):  
R. M. E. Parkhouse
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sucrose Density Gradient ◽  
Immunoglobulin M ◽  
Sucrose Density Gradient Centrifugation ◽  
Chain Synthesis

Immunoglobulin M (IgM) biosynthesis was studied with mouse plasma-cell tumour MOPC 104E as a model system. Cell suspensions prepared from solid tumours were incubated in vitro with [3H]leucine; the radioactivity incorporated into intracellular and secreted proteins was analysed by sucrose-density-gradient centrifugation and polyacrylamide-gel electrophoresis. The tumour secretes IgM and light chains. ‘Pulse–chase’ experiments indicated average secretion times of 1.5h for light chain and 2.5h for IgM. The order of disulphide-bond assembly within the cell was shown to be heavy chain+light chain → heavy chain–light chain intermediate → IgMs. The 7S subunit (IgMs) was polymerized into IgM just before or at the time of secretion. Measurements of heavy-chain/light-chain radioactivity ratios in intracellular HL and IgMs and secreted IgM demonstrated the existence of a light-chain pool participating in IgM biosynthesis. The size of the light-chain pool, together with analysis of clones isolated in vivo, suggested that the tumour contains cells in which light-chain synthesis is in excess of heavy-chain production.

scholarly journals Expression of the (recombinant) endogenous immunoglobulin heavy-chain locus requires the intronic matrix attachment regions.

1997 ◽  
Vol 17 (5) ◽  
pp. 2658-2668 ◽  
Author(s):  
A E Oancea ◽  
M Berru ◽  
M J Shulman
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Heavy Chain ◽  
Close Association ◽  
Immunoglobulin Heavy Chain ◽  
Regulatory Elements ◽  
Chain Gene ◽  
Matrix Attachment Regions ◽  
Heavy Chain Gene ◽  
Igh Locus

The elements which regulate gene expression have traditionally been identified by their effects on reporter genes which have been transfected into cell lines or animals. It is generally assumed that these elements have a comparable role in expression of the corresponding endogenous locus. Nevertheless, several studies of immunoglobulin heavy-chain (IgH) gene expression have reported that the requirements for expressing IgH-derived transgenes differ from the requirements for expression of the endogenous IgH locus. Thus, although expression of transgenes requires multiple elements from the J(H)-C mu intron--the E mu core enhancer, the matrix attachment regions (MARs) which flank E mu, and several switch-associated elements--B-cell lines in which expression of the endogenous heavy-chain gene is maintained at the normal level in the absence of these intronic elements have occasionally been reported. Gene targeting offers an alternative method for assessing regulatory elements, one in which the role of defined segments of endogenous genes can be evaluated in situ. We have applied this approach to the IgH locus of a hybridoma cell line, generating recombinants which bear predetermined modifications in the functional, endogenous mu heavy-chain gene. Our analysis indicates the following. (i) Ninety-eight percent of the expression of the recombinant endogenous mu gene depends on elements in the MAR-E mu-MAR segment. (ii) Expression of the recombinant mu gene depends strongly on the MARs of the J(H)-C mu intron but not on the adjoining E mu core enhancer and switch regions; because our recombinant cell lines bear only a single copy of the mu gene, our results indicate that mu expression is activated by MAR elements lying within that same mu transcription unit. (iii) The MAR segment includes at least one activating element in addition to those defined previously by the binding of presumptive activating proteins in the nuclear matrix. (iv) Close association of the MARs with the E mu enhancer is not required for MAR-stimulated expression. (v) The other MARs in the IgH locus do not in their normal context provide the requisite MAR function.

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