Biol3402 Exam 4 quick questions

just quick questions

How do cyclins activate CDKs?

Cyclins bind CDKs causing CDK conformation change that exposes T-loop.

What is the CDK T-loop and how is it regulated?

The T-loop contains a threonine that, when exposed by cyclin binding to CDK, gets exposed and can be phosphorylated by CAK. This CAK phos event increases CDK binding affinity for cyclin and other substrates, thereby increasing CDK activity.

Describe the series of events that lead from G0 → entry into S phase

Mitogen → cyclin D accumulation → out-competes INK for CDK4/6 → cyclin D/CDK4 initiates Rb phosphorylation that reduces E2F transcriptional repression → partially active E2F induces cyclin A and E transcription → cyclin E/CDK2 complete Rb phosphorylation leading to full lift of E2F transcriptional repression → E2F transcription of genes (e.g. cyclin A) that are necessary for S phase entry.

Describe how each technique can provide information about the cell cycle protein blot, luciferase assay, immunoprecipitation, in vitro kinase assay.

• Protein blot = protein accumulation and posttranslational modifications

• Luciferase assay = promoter activation

• IP = protein-protein interactions

• Kinase ability to phosphorylate a substrate

What experiment could be done with FAC’d cells that would provide insight into cell cycle regulation?

What leads to the start of cyclin A transcription in G1?

cycD/cdk4/6 and cycE/cdk2 phosphorylation of Rb and subsequent activation of E2F.

Describe gel shift experiment, which is used to show DNA protein interaction.

Gel shift = protein + label DNA run on non-denaturing gel and imaged. If protein binds DNA, will see reduced mobility in gel.

Describe the cyclin A feedback mechanism that helps drive the cell cycle forward. How does this mechanism help drive the cell cycle forward?

cycA/cdk2 blocks E2F transcriptional activity, thereby turning off cycA transcription. Also turns off cycE transcription and other genes important for S phase. Protein activity is phase limited to allow the cells to move forward once those phase limited protein functions are done.

Describe the role of cyclin A/CDK2 in DNA synthesis

cycA/cdk2 phosphorylates cdt1, cdc6 marking for degradation and phosphorylates ORC to release from DNA so that another pre-replication complex cannot be established (e.g. only one round of DNA replication per cell cycle). (cdt1 and cdc6 recruit helicase complex (MCM) to ORI where the ORC is bound). ORC marks ORI.

What do cyclin E/CDK2 and cyclin A/CDK2 phosphorylation of E2f and cyclin/CDK phosphorylation of Cdt1 indicate about the regulation of CDKs substrate specificity and function?

The cyclin dictates the substrate specificity and function of the cyc/CDK complex. - CycE/Cdk2 induces E2F, cycA/Cdk2 inhibits E2F; cycB/Cdk1 does not phosphorylate ORC, cycA/cdk1 can phosphorylate ORC.

Describe ChIP experiment, which is used to show DNA-protein interaction.

ChIP - formaldehyde fix cells to preserve transcription factor binding to DNA, sonicate DNA into small fragments, use antibody to IP protein-DNA complex, use PCR to amplify the protein bound DNA.

Describe the experiment used to show that a protein binds and phosphorylates it substrate.

In vitro kinase + IP + western for shifted phosphorylated protein.

What is some evidence that cyclin A/CDK promotes G2/M transition?

What role does Cdc25 play in G2/M transition?

What role does Wee1 play in G2/M transition?

Describe the feedback mechanism that amplifies cyclin B/CDK1 activity in M phase.

What are two chromosome regulatory functions of Cyclin B/CDK1 in M phase?

How does APC promote mitosis?

What are some physiological effects of cancer?

Define the stages of cancer.

• In situ: pre-cancerous, has not breached basement membrane

• Localized: primary site

• Regional : nearby tissues, lymph nodes

• Distant: distant tissues

Define the grades of cancer.

• G1: differentiated

• G2: moderately differentiated

• G3: poorly differentiated

• G4: undifferentiated

What is the difference between a tumor and cancer?

Tumors are proliferating cells that can be benign or malignant. Cancer cells are malignant.

What is the difference between benign and malignant?

Benign tumor is confined, malignant tumor breaches the basement membrane and spreads

What are the types of cancers (e.g. based on tissue origin)?

Carcinoma (epithelial), sarcoma (connective), lymphoma (lymph nodes), leukemia (marrow), myeloma (plasma)

Which cancer is more aggressive - T4N0M0, Gleason 3+4 or T4N1M1, Gleason 3+4 and why?

• T4N1M1, Gleason 7 because while both tumors are moderately/poorly differentiated (Gleason 7), this one has spread beyond the primary site to neighboring tissue (T4) and lymph nodes (N1), distant site (e.g. bones) (M1)