Dulic_guide_answers - Guide for Dulic et al. (1994)...

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Guide for Dulic et al. (1994) p53-Dependent inhibition of cyclin-dependent kinase activities in human fibroblasts during radiation-induced G1 arrest. Cell 76:1013-1023. These questions are intended to help you read the paper in an inquisitive fashion and to help you focus on some of the issues you should understand. Please cover each of these questions during your reading and studying. Please submit the answers to 5 of these questions for your homework assignment. 1. The authors use synchronized cell populations in their experiments. How are cells synchronized in this paper? Human fibroblasts were synchronized in G1 by allowing them to grow to confluency. As the cells contact one another in the culture dish, cell growth is inhibited. These growth- arrested cells were then replated at a lower density on new plates, allowing growth to resume. 2. How do Dulic et al. demonstrate that ionizing irradiation (IR) blocks entry into S phase? How do they measure S phase progression (see p. 989, MBOC)? Progression into S was monitored by labeling the cells with 3H-thymidine and then counting the number of radioactive nuclei after autoradiography of the cells (see p. 989, MBOC). When cells were treated with gamma rays, the number of radioactive nuclei diminished markedly indicating that the gamma rays were blocking DNA replication (see Figure 1 A). 3. The authors find that IR inhibits the kinase activity of immunoprecipitated cyclin A and cyclin E -cdk complexes as assayed by phosphorylation of histone H1 in vitro (see Figure 1B). However, the mechanism of inhibition appeared to be distinct for the two complexes. How does IR appear to affect cyclinA-cdk activity (Fig 1C)? It appears that IR inhibits the synthesis of cyclin A since the steady state level that is detected by western blotting is notably less than normal when the cells are irradiated (Figure 1C). By contrast, the western blot analysis does not show any change in the
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This note was uploaded on 10/19/2009 for the course B M B 460 taught by Professor Hanna-rose,w during the Spring '08 term at Pennsylvania State University, University Park.

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Dulic_guide_answers - Guide for Dulic et al. (1994)...

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