1.  Consider this situation: Cells need to control the concentration of a molecule called X. The synthesis of X is regulated by an enzyme, Xase. In order to be active, Xase needs another molecule, Y, which itself is regulated by inhibiting the activity of an enzyme, UnYase. If the concentration of all of these molecules is controlled by how quickly their mRNAs are degraded using a protein that works just like the Iron-Response Binding Protein, illustrate how their 3’ UTRs would look when molecule X is needed by the cell. (8 points)

X, Xase, and Y are all needed. UnYase is not. Therefore, the 3’ UTR of X, Xase, and Y mRNAs will have their stem-loops covered by binding to the protein, thus protecting them from degredation. UnYase mRNA will not have its stem-loops bound, and nucleases will degrade it.
 

2. One substrate of the cyclic AMP dependent protein kinase (protein kinase A) is glycogen synthase. Outline all the molecular events that lead to phosphorylation of this enzyme following hormonal stimulation of the cell in which it is found. (8 pts)

Hormone binds G-linked receptor.
Receptor cytoplasmic domain changes shape.
It interacts with the G protein causing the beta/gamma subunits to release the alpha subunit.
The alpha subunit now replaces GDP with GTP, becoming active.
The active alpha subunit binds to adenylyl cyclase, activating it.
Adenylyl cyclase catalyzes synthsis of cyclic AMP.
Cyclic amp binds to regulatory subunits of protein kinase A.
They release the catalytic subunits which can then bind substrates and phosphorylate them.
Glycogen synthase is one such substrate.
 

3. Calcium is often called a second messenger in cells. How does a cell increase calcium concentrations in the cytoplasm when needed? (8 pts)

Hormone binds and activates a receptor.
Receptor causes a Gq subunit to release from the beta/gamma subunits and activate phospholipase C.
Phospholipase C cleaves PIP2 into DAG (remains in the membrane) and IP3.
IP3 moves to the endoplasmic reticulum and causes the opening of a calcium channel.
Calcium is released into the cytoplasm.
The same calcium channel moves to the cell membrane and binds to and activates a membrane calcium channel, causing influx of calcium into the cytoplasm from the extracellular environment.
 
 

4. How does a receptor tyrosine kinase respond when bound by a signaling hormone? (Just discuss the receptor itself, not further downstream events) (8 pts)

Binding of the ligand to two monomeric RTKs causes them to dimerize. Then each monomeric unit of the dimer catalyzes attachment of a phosphate to its’ partner’s activation lip at a tyrosine. This further activates the tyrosine kinase activity of each monomer and causes each to attach further phosphates to tyrosines in the cytoplasmic region of each RTK.
 
 

5. Define the following terms. (3 pts each)

A. SH2 domain
Region of a signaling protein that attaches to phosphorylated tyrosines on the target protein (activated RTK)
 

B. SH3 domain
Region of a protein that is associated with the signaling cascaded mediated through an SH2 domain. Sometimes the SH2 domain is in the same protein as the SH3 domain, sometimes they are in separate, interacting proteins. The SH3 domain directly binds to other proteins high in proline content.

C. a GAP
A GTPase activating protein. It binds to an active, GTP-bound G protein and helps accelerate the GTPase activity of that G protein.

D. a GEF

A Guanine nucleotide exchange factor. It binds a GDP-bound inactive G protein causing GDP to leave and allowing GTP then to enter to reactive the G protein.
 

6. Outline the steps by which the cell-cycle is arrested during mid G1 in response to DNA damage. This involves two pathways, both of which are started by the ATM kinase. Be sure you include both pathways in your answer. (10 pts)

One pathway involves a signal to ATM/R that causes phosphorylation of mdm-2, causing it to be unable to remain bound to p53. p53, further stabilized by additional phosphorylations upon itself by other kinases, then activates the transcription both of itself and of p21. p21 binds to cyclin-cdk complexes, preventing them from phosphorylating their targets, thus preventing cell cycle progression.

A second pathway involves a signal to ATM/R which phosphorylates either chk1 or chk2 kinases. They then phosphorylate cdc25A phosphatase, inhibiting its activity. It would normally, if active, remove critical phosphates from cyclin-cdk complexes allowing cell-cycle progression. By preventing this, cell cycle is arreste
 

7. Below are six proteins that play an important role in regulating programmed cell death (apoptosis). Identify whether they are pro-apoptotic or anti-apoptotic. (12 pts)
 

Caspase 9   ___pro___________________

Bax ____pro______________________

14-3-3 ___anti_______________________

Non-phosphorylated Bad _____pro________________

Bcl-2 _____anti______________________

Akt kinase ______anti_____________________
 
 

8. How do scientists use normal mouse cells to identify human cancer-related genes? (6 pts)

Human tumor cell DNA extracted and used to transfect normal mouse cell monolayer.
Foci form, are picked, and DNA extracted from them and used to transfect normal mouse cell monolayer again.
Foci form again and the DNA is extracted and cloned into a phage library
Phage library used to infect bacterial cells, forming plaques.
Plaques are replica plated onto filter papers.
Filters are treated and potential hybrids formed between the DNA in the plaques and the ALU probe (identifies presence of human DNA sequences).
Positive plaques are thus identified and the DNA from them used to identify potential human oncogenes nearby the ALU sequences.
 

9. Name two ways that a proto-oncogene can become an oncogene. (6 pts)

Several possibilities:

Spontaneous mutation
Retroviral transduction
Insertional mutagenesis
Chromosomal translocation
Gene amplification
 
 

10. How do the following DNA tumor viruses inactivate tumor-suppressor proteins in order to allow progression through the cell cycle? (9 pts)

A. SV40
T antigen of SV40 binds to both p53 and to Rb tumor suppressor genes inactivating them.

B. Adenovirus
E1a protein of Adenovirus binds to Rb and E1b to p53, inactivating them.

C. HPV (Human Papilloma Virus)
E7 protein binds to Rb, inactivating it. E6 protein binds to p53 and targets its degredation.

11. Why are tumor suppressor genes also called loss-of-function or recessive oncogenes? (5 pts)

The tumor suppressor gene products produce a protein that plays a role in inhibiting cell cycle progression in some way, or preventing accumulation of mutations. Therefore, to knock out this protective function, both alleles of the gene for these proteins must be mutated, lost, or otherwise inactivated to see the effect that contributes to cancerous transformation of the cell.
 

12. Two new cancer medicines are called Gleevec and Avastin. Briefly describe how they work. (8 pts)

A. Gleevec
Gleevec inhibits the deregulated tyrosine kinase activity of mutant tyrosine kinases that participate in signals for cell division. The original such example of an unregulated tyrosine kinse was caused by the bcr-abl chromosomal translocation.
 

B. Avastin

Avastin inhibits the signal from tumor cells that causes angiogenesis (formation of new blood vessels). These new vessels are required for continued nutrients to be brought to the tumor cells. Inhibiting these new blood vessels “starves” the tumor, prohibiting its growth.