| The precise control of the key checkpoints of the cell cycle, such as G1/S, S/G2, G2/M and mitosis exit, ensures the eukaryotic cells to proliferate and divide in an orderly and programmed manner. The promoting molecules in cell cycle regulation include cyclins and CDKs (cyclin-dependent kinases). The CDKs are activated when they interact and bind with the proper regulating cyclins. It has been shown that different CDKs are responsible for the regulation of cell cycle progression by associating or dissociating the specific cyclins to phosphorylate or dephosphorylate specific substrates. CDIs (cyclin-dependent kinase inhibitors) are the negative effectors to CDKs. Moreover, some oncogenes and tumor suppressor genes are also important in cell cycle regulation; those include myc, ras, p53, p21 and p16. The abnormal expression of these genes may interfere with the normal transcription of certain genes associated with cell cycle progression and cell proliferation.It has been known for some time that the acetylation of N-termini of core histones in nucleosome is associated with gene activation. Upon extensive studies, it has been found that histone acetylation is important in cell cycle regulation. However, little is known about the mechanisms of this process up to date. We do not know what cell cycle-associated proteins are regulated by histone acetylation modification, especially at the checkpoints of G1/S, S/G2, G2/M and mitosis exit. Neither do we know about the manner in which histone acetylation regulates the expression of these genes. These issues are essential to the understanding of the nature of cell cycle control, and to elucidating the roles of the histone acetylation modification in cell cycle regulation in eukaryotes.In this thesis, we studied the mechanisms of histone acetylation in cell cycle regulation in Physarum polycephalum, a naturally synchronized slime mold. The results of this study confirmed that histone acetylation changed the expression of proteins related with the regulation of checkpoint conversion in cell cycle. Based on the data arising from the experiments in this thesis, a hypothesized model, which intends to explainthe mechanisms and relationship between histone acetylation and the expression of important cell cycle regulating factors in Physarum polycephalum. The main results and conclusions of this thesis are as follows.1. By treating the cells in S, G2 phase and prophase with histone deacetylase inhibitor TSA, and through the application of microscopic observation and Western-blotting, we demonstrated that histone acetylation modification played important roles in the cell cycle regulation in Physarum polycephalum, affecting the normal crossover of the checkpoints of S/G2, G2/M and mitosis exit.2. By using TSA treatment, RT-PCR and Western-blotting, we established that the histone acetylation modification changed the expression of various cell cycle-related factors (mRNA and protein expression) at different checkpoints. These factors included cyclin B1-like protein, P53-like protein, c-Fos-like protein, c-Jun-like protein and Ras-like protein in Physarum polycephalum.3. To investigate the functions of these factors, cells were treated with anti-c-Fos, anti-c-Jun and anti-Ras antibodies and examined microscopically for the cell cycle progression. The results indicated that c-Fos-like protein, c-Jun-like protein and Ras-like protein played important roles in checkpoint regulation in Physarum polycephalum. Western blot analysis showed that the c-Fos-like protein and c-Jun-like protein may act in cell cycle checkpoint by changing the cyclin B1-like protein and P53-like protein expression; and the Ras-like protein may act by changing the cyclin Bl-like protein , P53-like protein, c-Fos-like protein and c-Jun-like protein expression.4. Though TSA treatment and Western blotting, we demonstrated that acetylation of certain non-histones may also be associated with the regulation of checkpoints in Physarum polycephalum.5...
|
PDF Full Text Request