Gene expression and differentiation in antibody forming cells

Singer, Paul Allan (1978) Gene expression and differentiation in antibody forming cells. PhD thesis, University of Glasgow.

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Considerable interest is attached to the biochemical events which characterize B-lymphocyte gene expression and differentiation. The expression of Ig is central to these processes. Among the events which are important for an understanding of Ig expression are: 1) the mode of integration of H-chain V and C-region genes, 2) the expression of Ig H-chain genes during isotype diversification, 3) the mode of surface binding and signal transmission of the several receptor isotypes, 4) the pathways which regulate the expression of cell surface and secretory Ig, and 5) the mechanisms involved in amplification of Ig synthesis. Many of these processes can be studied at the level of the unstimulated B-lymphocyte. However, model systems of homogeneous B-lymphocyte cell lines have only recently been considered for detailed biochemical analyses. The aim of this thesis work was first to characterize a series of human lymphoma and lymphoblastoid cell lines with respect to Ig expression. It was hoped that these cell lines would be characteristic of a series of early stages in B-cell differentiation. Secondly, using these cell lines as model systems, an investigation was undertaken to determine the cell surface binding and biosynthetic pathway of cell surface associated Ig. Many of the lymphoma cell lines investigated were found to synthesize predominantly cell surface IgM (sIgM), with varying degrees of IgM secretion as well. One lymphoma cell line (U698M) coexpressed cell surface IgM and IgD. Another cell line (Raji) synthesized IgM, but secreted none, and deposited very little on the cell surface. The lymphoblastoid cell lines synthesized and secreted IgM, with very little surface deposition. One lymphoblastoid cell line (Bec-11) secreted IgG and retained a significant quantity of IgG on the cell surface. It was concluded that these cell lines were analogous in their Ig expression to many of the cell types described in B-cell ontogeny and during mitogen stimulation. SDS-PAGE analysis of cell surface and secretory IgM p-chains revealed a reproducible relationship in the molecular sizes of the isolated chains. Cell surface gamma-chains were found to be larger than either secretory or intracellular gamma-chains from IgM secreting cells. Cell surface IgG from Bec-11 cells was also found to have a larger gamma-chain than its secreted counterpart. The basis for the gamma-chain size differences was further investigated in light of previous reports of the same phenomenon, and of a current hypothesis that cell surface gamma-chains may have a C-terminal extra hydrophobic sequence for integral membrane binding. Evidence was obtained which strongly supports this hypothesis. Thus, the higher molecular weight gamma-chain (putative surface form) was shown by carboxypeptidase digestion to have a different C-terminal amino acid residue than secretory gamma-chain. This was the first actual correlation of a C-terminal difference with the higher molecular weight form of gamma-chain. A working hypothesis, that retention of the putative N-terminal precursor sequence on surface y-chains was involved in membrane binding, was also tested. This hypothesis was disproved on the basis of N-terminal microsequencing analysis (performed in collaboration with Dr. Donald Capra), which indicated the presence of unblocked V N-terminal sequences on both Daudi and Raji cell surface gamma-chains. However, evidence was obtained which suggested a less efficient processing of N-terminal precursors in nonsecretory cells. Further analysis of the Ig H-chains (and L-chains) from nonsecretory and secretory cells was carried out by comparative SDS-PAGE of the mature cellular products, and their nonglycosylated and cell-free synthesised counterparts.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: A R Williamson
Keywords: Biochemistry, Immunology
Date of Award: 1978
Depositing User: Enlighten Team
Unique ID: glathesis:1978-72804
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 11 Jun 2019 11:06
Last Modified: 11 Jun 2019 11:06

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