Question

1. You are studying a particular type of lung cancer, small-cell lung carcinoma. You are interested on driver mutations of this cancer, that is mutations that provide strong selection advantage and thus have strong potential in initiating tumorigenesis. You have access to primary tumor biopsies from patients, and healthy surrounding tissue: how you can best use these tissue samples to identify mutations underlying this particular type of lung cancer?

2. The mutation you isolated has not been characterized before; how can the crystal structure of the protein bound on DNA help you understand how the mutation affects the function of the protein? What are some common mutations of the p53 protein that drive cancers?

3. You believe DNA binding is affected by the identified mutation, and your next goal is to characterize the functional perturbations of the mutant p53. a. Describe an in vitro approach to study the function of the mutant protein. b. Describe an experimental approach using cultured cells that can illuminate the altered function of the mutant protein

4. You find in #3 that the expression levels of certain miRNAs are changed as a result of the p53 mutation. a. What experimental approach can reveal how altered miRNA levels change Ago/miRNA targeting of mRNAs at the transcriptome wide level? b. What experimental approach can quantify altered translation levels for the mRNAs identified in a)?

Answer 1

Question 1.

Two things can be proposed

a. to perform DNA sequencing of both tissue samples and identify the mutations.

b. Continuous cell culture approach. Revive the cells from both biopsies in the cell culture. then identify pathways which affected in the cancerous cells. This can be done in two ways

• Lyse the normal cells and tumor cells; identify the metabolites in both types of cells by doing NMR and mass spectroscopy. Analyze the results for an increase or decrease of metabolite in both the cells and trace the pathways involved in their biogenesis or degradation.
• Isolate the mRNA from both cells and perform Microarray. Results can be traced for the increase or decrease in the expression of a protein or enzymes involved in a particular pathway.

Question 2.

Interaction of the protein to the DNA leads to the formation of functional for of protein. How it is possible? Let us consider protein is functional in its tetramer form. Binding of monomer to the DNA will cause structural changes in the monomer which allows the binding of other units of monomer to form functional tetramer form. The crystal form of the protein along with DNA will help us to understand does binding with mutant DNA will lead to the formation of functional form?

Mutations of p53 that drive cancer are TP53, CDKN2A (p16), DPC4, BRCA2, etc.

Question 3.

In vitro approach, one can check for the gain in function or loss of function of a particular enzyme. If protein is not the enzyme then its interactions with the known partner can be studied.

If protein is an enzyme then study its function by using its natural substrate and measure the amount of product it is formed. If it is not an enzyme then perform Forster resonance energy transfer (FRET) analysis to know the energy transfer between to interacting partner. In this technique, the interacting protein will be tagged with fluorescent donor and acceptor molecules. If protein will interact (they will come together) and hence energy transfer will be there between two tagger molecules, if proteins will not interact then there will be no transfer.

In the cell-based assay,

Fluroscent tag like GFP can be attached with the protien under question and monitor the fluroscent tag of protien invovled in functions by using in vivo cell imaging technique.

Question 4

To know the transcription wide level, an HT-SELEX experiment can be performed. Polysome analysis can be performed to know the alteration in translation levels of mRNA.