ANSWERS Functional Genomics: Exploring the Genetic Model System C. elegans. Part I. Forward and reverse genetics for insight into longevity. (20 pts) A. Watch this 5-minute video clip by scientist Cynthia Kenyon on a long-lived mutant in C. elegans (http://www.youtube.com/watch?v=5snTlCWqclY) and read Dr. Kenyon’s first-person history of the long-lived mutants (http://kenyonlab.ucsf.edu/html/history.html) (20 pts) 1. Based on the graph shown in the video, what is the maximum number of days that the daf-2 mutant can live? (1 pt) 70 days, or ~70 days 2. Compared to the normal worms, the daf-2 mutant can live _2___ times as long. (fill in the blank with a number) (1 pt) 3. In the last sentence of the video clip, Dr. Kenyon states her research goal. Assume that the daf-2 gene has just been cloned and nothing other than the gene sequence and mutant phenotype are known. Based on what you have learned in this class, what are 3 likely questions that Dr. Kenyon might ask next about the daf-2 gene or protein, and what types of experiments could she carry out to answer those questions? Your answer should be based on the methods and approaches that we have discussed in this course and should be realistically feasible. (There are a variety of possible correct answers.) (9 pts) Some possible answers: 1) What is the gene expression pattern of daf-2? The experiments to answer this are northern blots, qPCR, in situ hybridization and/or transcriptional reporter genes, examining expression at different developmental stages and throughout the body of C. elegans. 2) What is the protein expression pattern of daf-2 protein? Some possible experiments to answer this are western blots, immunohistochemistry and/or translational reporter genes, to examining protein expression patterns at different developmental stages and throughout the body of C. elegans. 3) What proteins interact with the daf-2 protein? Use the yeast two-hybrid assay to screen a C. elegans cDNA library to find plasmids in the library that carry C. elegans genes that encode daf-2-interacting proteins. If you have an antibody against the daf-2 protein, you can use co-immunoprecipitation to pull down daf-2 along with associated proteins out of a protein extract of C.elegans, followed by mass spectrometry to identify the associated proteins. Other possible answers: 1) What would happen if daf-2 is ectopically expressed? Construct a daf-2 transgene to express in C. elegans and introduce the construct into C. elegans by microinjection. 2) What genes are expressed in the daf-2 mutant compared to the wild-type C. elegans? Use microarrays or RNA-seq to identify all of the genes expressed in daf-2 vs. wild-type during different stages of development. 4. Please read Dr. Kenyon’s story on the discovery of long-lived mutants on her website here: http://kenyonlab.ucsf.edu/html/history.html. Was the research on daf-2 an example of forward genetics or reverse genetics? (2 pts) Forward 5. As Dr. Kenyon mentions in her webpage story, the daf-2 gene was cloned by Dr. Ruvkun’s lab. What kind of protein does the daf-2 gene encode? The correct answer can be found in her story. Alternatively, go to the C. elegans database website(www.wormbase.org). If you visit the C. elegans database, you can search for “daf-2” using the “search for a gene” tool in the upper right hand corner, which will take you to an information page about daf-2. (1 pt) A receptor tyrosine kinase (which is the C. elegans insulin/IGF receptor). 6. In the story on Dr. Kenyon’s webpage, what is the name of a second daf mutant that affects aging, and what was Dr. Kenyon’s nickname for this gene? (2 pt) daf-16 (1 pt) nickname is “sweet sixteen” (1 pt) 7. What is the genetic relationship of this second gene to daf-2? That is, does one gene control the other? Please include in your answer a simple diagram that shows the genetic pathway/relationship of these two genes as reported by Dr. Kenyon, using an arrow or inhibition sign between them. (2 pts) daf-2 regulates daf-16, or daf-2 negatively regulates daf-16 (more detail is fine) Simple diagram showing their genetic relationship: daf-2 -----| daf-16 9. What was the general approach that Dr. Ruvkun’s lab used to clone the daf-2 gene, as published by his lab? Please cite the full reference where you found the answer. (2 pts) The general approach is called “genetic mapping”, “linkage mapping” or “map-based cloning”, etc. The expected citation is the original cloning paper that is cited in Dr. Kenyon’s first person history: Kimura et al. (1997) daf-2, an Insulin Receptor-Like Gene That Regulates Longevity and Diapause in Caenorhabditis elegans. Science 277: 942-946. Other sources, such as a review article, could be correct as well. B. Examine the research paper by Hamilton et al. (2005) to answer the following questions regarding Hamilton et al.’s research. The pdf of Hamilton et al. is posted on Blackboard in the homework assignment folder under Projects. (17 pts) 1. What was the goal of this paper, and what was the general experimental approach that was taken? (2 pts) Minimal answer: The goal was to identify/screen for longevity genes/mutants in C. elegans (1 pt) and the experimental approach was to carry out a systematic (genome-wide) RNAi screen (1 pt) 2. How many clones were tested in this study and what approximate percentage of the genome does this represent? (2 pts) 16,475 clones were tested (1 pt), representing 16,475 clones/~19,000 genes in C. elegans = ~86.7% of the genes in the genome (“>80%” is acceptable) 3. How many genes did the authors identify? (1 pt) 89 (90 is acceptable) 4. The efficacy of the experiment was validated by the identification of genes that had been previously known to be involved in longevity, including daf-2. Give the names of two such genes other than daf-2. (2 pts) age-1 and akt-15. Of the list of genes identified in this study, what was the most prevalent functional group of these genes (other than the category of “unknown”)? (2 pts) Metabolism (“Signaling” is acceptable, since metabolism can be separated into Electron Transport Chain, TCA, etc) 6. The authors reported that orthologs exist for many of these listed genes. For what 3 organisms did they report these orthologs? (1.5 pts) fly, mouse, human 7. According to the paper, what was the authors’ stated