Thymidine kinase mRNA and protein levels during myogenic with drawal from the cell cycle : identification of an mRNA-independent regulatory mechanism Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/w9505393h

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  • Replication associated protein and enzyme activity levels increase as cells enter S-phase of the cell cycle and diminish as cells leave S-phase. Accordingly, replication associated functions decrease as myoblasts withdraw from the cell cycle to terminally differentiate. In an effort to understand signals effecting growth associated expression of genes, the molecular mechanism controlling declining thymidine kinase (TK) activity levels during myogenic withdrawal from the cell cycle was investigated. Initially, the hypothesis that TK was regulated at the level of mRNA was investigated both in vivo and in an in vitro myoblast cell culture system. Qualitatively, TK mRNA declined by a transcriptional mechanism. However, quantitative comparison of the decline in TK mRNA and TK activity revealed TK activity was regulated by a mRNA-independent mechanism. Consequently, the hypothesis that TK activity was regulated by a posttranslational mechanism was tested. Antibodies against TK protein were derived and used to demonstrate, via immunoblot and immunoprecipitation experiments, the existence of a translational or protein degradational mechanism. The possible contribution of posttranslational modulation of TK activity could not be rigorously eliminated. A second approach to understanding the mechanism of decline of TK activity during myogenic withdrawal from the cell cycle involved further localization of intragenic cis-acting regulatory elements. Regulation of TK activity was monitored in myoblasts transformed with intron deletion mutants of TK. Introns were inconsequential to regulation of TK activity. Thus, cis-acting regulatory elements mediating the decline in TK activity were within the protein coding region, consistent with the translational or protein degradational level of regulation. Quantitative evaluation of TK mRNA regulation in myoblasts transformed with promoter switch, 3' replacement, and intron deletion mutants also localized cis-acting elements mediating the transcriptional decline in TK mRNA to the protein coding region. However, the controversy surrounding the nature of the heterologous promoters used, the smallfold and variable decline in TK mRNA, the possibility of redundant control elements, and the unusual location of the transcriptional regulatory element cast doubt on this conclusion. Two general mechanisms for controlling TK mRNA levels were proposed. The available set of intron deletion mutants was used to test the popular hypothesis that introns are essential for expression of mRNA. Quantitative evaluation of TK mRNA expression in mouse fibroblasts transformed with full length TK genes or intron deletion mutants revealed no significant difference in expression. Thus introns were inconsequential to expression of TK mRNA in fibroblasts.
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