Myogenic differentiation of muscle cells in culture is characterized by changes in morphology and in pattern of gene expression. When the myoblasts in culture are induced to diffrentiate either by cell to cell contact or by serum-starvation, a vast array of muscle tissue-specific genes including α-actin, are activated and is accompanied by a concomitant down-regulation of non-muscle genes such as, β- and γ-actins, tubulins etc.. The coordinate activation of muscle-specific genes is suggested to be mediated by cis-acting regulatory sequences in the muscle gene-promoters and muscle-tissue-specific DNA-binding proteins belonging to the MyoD class of regulators. But the mechanism behind the repression of non-muscle gene expression during differentiation has not yet been well understood. To date, no consensus has been achieved on the mechanism governing the down-regulation of β- actin gene, and no information is available on the regulation of -y-actin gene during muscle differentiation. The results from the present study showed that during differentiation of BC^3H1 cells the β- and γ-actins genes were down-regulated to ≈25% of their initial levels in undifferentiated cells. Measurement of half-life during differentiation indicated that the half-lives of both β- and γ-actins decreased to ≈25% of their original levels in myoblasts. These results suggest that changes in mRNA stability play an important role in the down-regulation of non-muscle actin genes. Second messengers and oncogenes are known to block the differentiation program of muscle cells. In the present study cAMP and ElA were observed to inhibit the down-regulation of β- and γ-actin genes in BC^3H1 cells. In both cAMP-and E1A- treated cells the β- and γ-actin mRNAs were found to have a higher half-life than the untreated differentiated BC^3H1 cells. This observation also suggests that mRNA stability might play an important role in the regulation of β- and γ-actin gene expression. The muscle-specific α-actin is activated by cell-cell contact and serum-starvation. Results in the present study suggested that cAMP was able to inhibit the activation of α-actin expression mediated by serum-starvation while it had no significant effect on the signal mediated by cell-cell contact. It is hypothesized that the two signals mediating a-actin activation might follow different intracellular signalling pathways. The effects of cAMP and E1A on the expression of muscle-specific and non-muscle actins could be a direct primary event or might be an indirect secondary event, mediated by other intracellular factors such as myogenin. The results showed that cAMP did not block the transcription of the myogenin while secondary evidences suggested that cAMP might negatively-regulate myogenin at a point downstream of transcription. E1A was observed to block the expression of myogenin gene suggesting that E1A might be mediating its effect through myogenin. Because the muscle-specific(α) and non-muscle(β- and γ-) isoforms were expressed both in the presence and in the absence of myogenin, myogenin's role in the regulation of actin genes is unclear. / Thesis / Master of Science (MS)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/22431 |
| Date | 10 1900 |
| Creators | Muthuswamy, Senthil Kumar |
| Contributors | Lee, Raymond W. H., Biology |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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