1 7.03 PROBLEM SET 5 KEY BASED ON LECTURES 20-25 DUE BEFORE 5PM ON WEDNESDAY, NOVEMBER 15 SUBMIT ANSWERS DURING RECITATION OR PLACE IN BOX OUTSIDE OF THE BIOLOGY EDUCATION OFFICE 1) Genetic pathways in eukaryotes often are investigated using gene fusions. This approach could be used in yeast to study regulation of a detoxification gene, DTX1. This gene encodes an enzyme that neutralizes benzene, which is a known carcinogen. To investigate DTX1 regulation, you made a gene fusion (PDTX1 - LacZ) that consists of the cis regulatory region of DTX1 and the coding region of LacZ. When integrated into the yeast genome, this gene fusion shows wild-type expression. In addition, you isolated two recessive loss-of-function mutations, dtx2- and dtx3-, which show un-inducible gene fusion expression. Your analysis shows that the dtx2- and dtx3- mutations reside in different genes, are not linked to each other, and are not linked to the gene fusion. a) Diagram three possible models that illustrate the wild-type regulatory relationships among DTX2 and DTX3 and PDTX1 - LacZ. These are the three simplest models.2 b) You have access to a dominant allele of the DTX2 gene, dtx2-D, which causes constitutive expression of PDTX1 - LacZ. Describe the experiment you would perform to distinguish between two of the three models diagrammed in part (a). Show all crosses, the resulting tetrads, and how each result should be interpreted. Cross dtx2D to dtx3-. The resulting diploid will be heterozygous for both dtx2-D and dtx3-. Sporulate this diploid to get the following tetrads listed below. Genotypes PD 2 uninducible, 2 constitutive NPD 2 wild-type, 2 double mutant (either constitutive or uninducible) TT 1 uninducible, 1 constitutive, 1 wild-type, 1 double mutant (either constitutive or uninducible) Take the double mutant spore from the NPD tetrad. - If PDTX1-LacZ is constitutively expressed in the double mutant, then discard model 1. - If PDTX2-LacZ is uninducible, then discard model 2. - This technique does not address parallel pathway models. c) Now you want to investigate the mechanism by which DTX2 and DTX3 act in the pathway. To determine if DTX2 and DTX3 can bind to each other, you decide to perform a yeast-two-hybrid assay. Which cis-regulatory regions would you put upstream of LacZ? You would place the promoter from the Gal1 sequence in front of LacZ. The UAS sequence is essential as it is the sequence in the Gal1 promoter to which the DNA binding domain of Gal4 binds. Suppose you engineer the protein fusion genes DTX2 : AD and DTX3 : DB on a selectable plasmid. What results from the yeast-two-hybrid assay would show that DTX2 and DTX3 interact/bind directly to each other? Complete the chart below and include the necessary controls. Assay Yeast strain Reporter gene expression (expected) Control 1 Pgal1::LacZ None Control 2 DTX2 : AD / Pgal1::LacZ None Control 3 DTX3 : DB / Pgal1::LacZ None Experiment 1 (assume no direct interaction) Pgal1::LacZ + DTX2:AD + DTX3:DB None Experiment 2 (assume direct interaction) Pgal1::LacZ + DTX2:AD + DTX3:DB ++++3 The controls outlined in the table above are essential to do this experiment. There are other possible controls. For example, switch AD and DB so that DTX2 and DTX3 are attached to different domains and use this to confirm the interaction again. d) What if one of the controls listed above shows reporter gene expression? For each control, suggest a possibility as to what is occurring in the cell if reporter gene expression is observed. There are several possible answers for “part d”. The purpose of the question was to get you to think about how the assay works and why controls are essential. Control 1 There is something activating expression in a strain with only the fusion gene. This strain will not be useful; it is necessary to integrate the reporter gene somewhere else, or use a different plasmid. Control 2 DTX2:AD is activating reporter gene expression without the DNA binding domain of Gal4. DTX2 may bind directly to DNA close enough to the reporter fusion gene to activate its expression. Control 3 DTX3:BD is recruiting RNA Polymerase or is recruiting other factors that promote LacZ expression without the activation domain. e) Based on the results from the yeast-two-hybrid assay, what can be concluded about your models in part a? If the proteins do interact with each other in the yeast two hybrid assay, it suggests that the proteins may interact with each other when they promote DTX1 expression. If the proteins interact, it is strong evidence that they may act in a pathway in series instead of in parallel. If you find they do not interact, new information will be necessary.4 2) Genes in the body that suppress the development of cancer are known as tumor suppressor genes. Due to its role in the repair of double-strand breaks in DNA, REP1 is regarded as a tumor suppressor gene. Individuals with loss-of-function mutations in both REP1 alleles show a much greater risk for developing some cancers. After brainstorming with colleagues, it is decided that the role of REP1 in cancer development could be investigated effectively using genetically modified mice. a) In order to obtain an effective mouse model to study the role of REP1 in cancer development, what genotype would you generate? Explain. REP1-/- To model this loss-of-function mutation, we must generate a mouse that lacks two functional copies of REP1. Based on REP1’s known role as a tumor suppressor, we would expect REP1-/- mice to show a greater risk of developing certain cancers. (NOTE: In certain cases, generating a homozygous mutant as we’ve done here is not possible. If a gene is serving an essential role, then losing the function of both of its alleles will produce unviable offspring. Here, since we are told that REP1-/- individuals are at a greater risk of developing cancers, we know that homozygous REP1 mutants are viable.) b) Would pronuclear injection or gene targeting techniques be required to construct the desired mouse model? Explain. Gene targeting. You hypothesize that the increased risk of cancer is due to two inactive versions of REP1. To model this situation in a mouse, you need to knock-out both functional copies of the REP1 gene. Gene targeting is the only method to do this.5 c) Exon 2 is essential for the tumor-suppressor activity of the REP1 gene (illustrated below). Draw the DNA construct
View Full Document
Unlocking...