MCDB 135 Midterm 2 Key

1A. CIf you wish to look at expression of an IEG, you must use a reporter that is under the control of a promoter of an IEG, such as cyclin E

  1. Lane 5 shows that an 80 kDa protein co precipitates with MegaT protein (200K). 5 pts

This is likely to be specific because it is 

  • observed only in the MV40 infected cells (lane 5) and not in mock (lane 3) or dMV40 cells (lane 7) 1.5pts
  • the control Ab (lanes 2, 4 and 6) do not show either the 82 or 200 kDa proteins. We expect to see the IgG band (heavy chain) in all lanes because all lanes have antibody in them. 5pts

The lower, unlabeled band in all lanes is either a spurious, sticky protein that binds any IgG (or perhaps they the antibody proteins). 0.5 pt (no need to say both possibilities)

  1. The experiment must directly test the hypothesis that the 80 kDa protein is in fact UTE (as opposed to some other protein that happens to be the same MW). There are multiple answers possible – need only 1 for full credit.

Possibility #1: Conduct a reciprocal co-IP. Use the anti-UTE antibody under the same situations as shown in question 2. We expect to see the 80kD protein (UTE) in lanes 3,5 and 7 because it is a cellular protein and present in all samples. We expect to see MegaT (200 kDa) co-precipitating with UTE only when cells are infected with wt virus (lane 5).

Possibility #2: Repeat the same experiment as show in question 2, but instead of staining the gel, transfer the proteins to a membrane and probe with anti-UTE. The 80 kDa band should label (present only in lane 5).

Possibility #3: Conduct the co-IPs as in question 2, then directly sequence the precipitated proteins using tandem mass spectrophotometry to see if it matches the UTE sequence. This is okay, but could be misleading. It might be better to cut the 80K band out of the gel shown in question 2 and sequence it.

4A. Need only 1 answer.

Since UTE is a ts regulating entry into S phase and UTE is inhibiting X…

The most likely target genes of X are either those encoding transcription factors that promote expression of driver cell cycle proteins or the drivers of the cell cycle themselves (okay to say just one or the other). 

Another possibility is that X directly or indirectly represses expression of genes encoding inhibitors of cell cycle progression.

4B. ChIP-SEQ is a good approach to identify target genes. (1 pt) 

Isolate cross-linked DNA-protein complexes from cells using an antibody directed againstprotein X (the “chromatin immunoprecipitation” part). Then, reverse the cross linker and sequence the bound DNA. Identify the genes that belong to the sequence. (3 pt)

You should do this under conditions where protein X is likely to be in the nucleus and actively bound to target genes. For example, when UTE is phosphorylated and has released X.

5A. Need only 1 answer.

This could be due to epigenetic marking – perhaps there has been a chromatin remodeling event via histone modification or direct DNA methylation, for example, in the region of the Mal2 regulatory region that shuts down expression.

A formal possibility is that there is a lof mutation in a TF that promotes mal2 expression or a GOF mutation in a TF that inhibits mal2 expression.

5B. If Mal2 is a TSG, then a lof is oncogenic. The most likely explanation for how a lof mutation can be heterozygous dominant is via a dominant negative mutation (a missense mutation in the coding region). Perhaps Mal2 works as an oligomer and the mutation causes it to have a conformation that “poisons” the oligomer, even if it contains a copy of the wt version of Mal2.

5C. The blots suggest that Mal2 levels accumulate over time and then eventually CDKi levels increase. This indicates that the DNA-damaging irradiation is inducing a cell cycle arrest response. It is possible, but not tested, that Mal2 directly activates CDKi expression in response to DNA damage.

The graph indicates that Mal2 is required for cell death; even though the cells have DNA damage, they do not die when Mal 2 is missing. The heterozygous cells do die, but not as well as the wt cells.

Together, these data suggest that DNA damage induces cell cycle arrest and then death andthat Mal2 is required for this.

5D. BAX should be induced at detectable levels by 10 hrs (definitely not at 0) and very high by 25 min, consistent with the graph in 5C. We expect to see a bit of a lag between CDKi and BAX expression.

5E. If a cell is unable to die in response to DNA damage or improper cell cycle regulation, this means that the cell has acquired the hallmark of avoiding apoptosis in addition to having mitogen-independent proliferation (which is caused by the Ras gof).

5F. For each, 1 pt for a method that makes sense and 2 pt for what is expected to happen over time.

Cyto c: localize expression using an antibody or specific stain for cyto C. We expect to see it move from the mitochondria to the cytosol over time as apoptosis occurs. Could be via direct imaging or cell fractionation followed by immunoblots (separate mitochondrial fraction from cytosolic fraction).

Caspase 9 – we expect caspase 9 to be activated; it will be activated by proteolysis and then cleave targets (such as PARP). Perform immunoblot analyses evaluating caspase 9 or PARP size over time – expect to see the protein get smaller (as it is cleaved). It is also possible to detect caspase 9 (and PARP cleavage) in cells using dyes and antibodies specific to cleaved products.

Note: When you carry out localization studies, it is always a good idea to evaluate proteins known to localize to a specific sub cellular locale as a positive control. For example, tubulin should not be in the mitochondria (it is a marker for “cytosol”) and the Cox4 protein should always be in the mitochondria, not the cytosol (it is a marker for “mitochondria”).

6A. Although most of the cells may exhibit hyperplasia (abnormal cell division), only a subset in the field of cells will accrue more oncogenic driver mutations over time that cause them to achieve additional hallmarks and to “cross the line” to form actual tumors.

6B. construct ii

6C. In order to faithfully “report” Ki29 expression, the endogenous Ki29 gene enhancer/promoter must be used. It will drive GFP expression only when the endogenous gene is also active. 

The other two constructs express GFP constitutively (or at the very least, would report “ubiquitin” gene activity, not Ki29 gene activity).

6D. YES. For two main reasons. First, Ki29 is a stem cell marker. Since telomerase is expressed in stem cells, it is possible that telomerase, also expressed in stem cells, is now expressed. Second, the cells that form tumors are likely to have acquired the hallmark of avoiding senescence and avoiding karyotypic crisis. In order to achieve the latter, they must solve the end replication problem, which they can do by expressing telomerase in order to maintain telomere length.

7A. Regardless of the method, we expect to see Hif1 protein at low, basal levels and localized in the cytoplasm initially. Then, as cells acquire the ability to induce angiogenesis, we expect to see Hif1 levels stabilize and increase, and that it moves into the nucleus. 

This could be due to hypoxia, or a gof mutation in Hif1, or a lof mutation in VHL. 

There are several ways to do this, but whatever method is chosen, it must reveal Hif1 protein(not RNA) over time, because localization of the protein is what matters here.

The best way is to monitor is in real time using a tagged Hif1 (engineer a transgene into the cells that uses the Hif1 enhancer/promoter to drive expression of a Hif1-GFP fusion protein). It has to be a tagged Hif1 in order to see where Hif1 is localized. GFP by itself will just stay in the cytoplasm.

You could also do immunohisto chemistry using an antibody against Hif1 as well.

Another method, though more cumbersome, is to isolate the tumor cells at various times, isolate nuclear and cytosolic protein fractions, then use immunoblot analyses to assess where the Hif1 protein is located (using an anti-Hif1 Ab).

7B. There are three basic ways – need only 1. Either way, the tumor has acquired the hallmark of being able to induce angiogenesis.

Possibility #1. The tumors are large enough that cells far from existing vasculature become hypoxic. This results in stabilization of Hif1 (it cannot be hydroxylated), which goes into the nucleus and causes expression of VEGF, stimulating angiogenesis.

Possibility #2. A mutation may have occurred in 

(i) The VEGF regulatory region that upregulated expression in a Hif1-independent manner.

(ii)

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