Post-transcriptional control of immunoglobulin synthesis

Laidlaw, Stewart A (1977) Post-transcriptional control of immunoglobulin synthesis. PhD thesis, University of Glasgow.

Abstract

Studies on the translation of mRNA of several secreted proteins and polypeptides in heterologous cell-free protein synthesizing systems have indicated that such secreted proteins are synthesized in vitro as precursor molecules. These precursors contain additional amino acid residues at their N-termini which appear to be rapidly removed in vivo. In cell-free systems, mRNA from Ig-secreting myeloma cells, which generally produce and secrete Ig H and L chains, has been shown to produce a higher MW precursor to L chain. Much less evidence exists for the presence of a precursor to H chains in vitro. Production of precursors to H and L chains of Ig was investigated in this project, using a recently developed cell- free system. The presence of two major polypeptide products of myeloma mRNA also allowed the relative effect of various inhibitors of protein synthesis on different mRNA's to be investigated. Cell-free translation of both polysomes and poly(A) RNA from several myeloma cell lines was investigated in this project. Ig H and L chains were identified as products of cell- free translation of Pl.17 polysomes in rabbit reticulocyte lysate by specific immunoprecipitation. More rigorous identification, involving both direct and indirect immunoprecipitation and comparison of producing and non-producing cell lines was carried out in a messenger- dependent lysate system. From these studies the following conclusions were drawn 1. In all cell lines investigated, Ig L chain was synthesized as a higher MW precursor, when produced in the cell-free system. In one cell line (5563) further characterisation by tryptic mapping showed the cell-free product to be related to the cellular product. This precursor also did not appear to have methionine as its N-terminal amino acid. 2. In all cell lines investigated, H chains appeared to be synthesized in cell-free systems with a lower apparent MW than the appropriate cellularly synthesized H chain. Inhibition of carbohydrate addition intracellularly mimicked this effect in one cell line (5563). The work in this project left unresolved the question of the existence of a precursor to H chains. Studies on the effect of inhibitors of initiation of protein synthesis on cell-free protein synthesis were carried out with Pl.17 mRNA. It was concluded that: 1. Ig H and L chain mRNA's were much more resistant to inhibition of initiation than other myeloma mRNA's. 2. Ig H chain mRNA was more resistant to inhibition of initiation than Ig L chain mRNA. Finally, an improved cell-free protein synthesizing system for the translation of polysomes was developed from the existing M.D.L. system by removal of endogenous amino acids.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: Alan Williamson
• Keywords: Molecular biology
• Date of Award: 1977
• Depositing User: Enlighten Team
• Unique ID: glathesis:1977-72802
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 11 Jun 2019 11:06
• Last Modified: 11 Jun 2019 11:06
• URI: https://theses.gla.ac.uk/id/eprint/72802