Cox, Angela; (1991) The stability of immunoglobulin messenger RNA in B cell lines. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

An important determinant of the level of protein produced from any particular gene is the cellular content of its messenger RNA, which in turn may be regulated at the levels of both synthesis and degradation. For example, as B cells differentiate, there is a large increase in the abundance of messenger RNA coding for immunoglobulin, which is not fully accounted for by an increase in the rate of gene transcription. The aim of this work has been to investigate the role of changes in messenger RNA stability in the regulation of immunoglobulin gene expression. Initial experiments used a model system, consisting of mouse fibroblast cells containing a high copy number recombinant human growth hormone gene, to develop a method for measuring RNA half-lives in mammalian cells. Pulse chase analysis was carried out using a ribonuclease protection assay to detect specific transcripts. This method was then used to investigate the rates of decay of immunoglobulin heavy chain RNA in two cell lines representing the resting B cell and the plasma cell stages of differentiation. It was found that heavy chain RNA had a significantly longer half- life in the cell line representing the plasma cell stage. This increase in half-life makes a significant contribution to the increase in heavy chain mRNA levels in these cells. To investigate factors which might control the stability of heavy chain RNA in plasma cells, a transfection system was employed to facilitate half-life measurements on a variety of mutated heavy chain RNA molecules. Heavy chains altered at their 5' and 3' ends were placed under the control of a heat shock promoter, and transfected into cells representing the plasma cell stage. This allowed a pulse of heavy chain mRNA to be generated upon treatment of the cells with elevated temperature, the decay of which could be monitored by Northern blot analysis. It was found that sequences at the 5' end of the molecule are important for its stability in cell lines representing the plasma cell stage.

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