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 1 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 PD NPD TT Genotypes 2 uninducible 2 constitutive 2 wild type 2 double mutant either constitutive or uninducible 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 twohybrid 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 Control 1 Pgal1 LacZ Reporter gene expression expected None Control 2 DTX2 AD Pgal1 LacZ None Control 3 DTX3 DB Pgal1 LacZ None Experiment 1 assume no direct interaction Experiment 2 assume direct interaction Pgal1 LacZ DTX2 AD DTX3 DB Pgal1 LacZ DTX2 AD DTX3 DB None 2 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 3 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 4 c Exon 2 is essential for the tumor suppressor activity of the REP1 gene illustrated below Draw the DNA construct you would use to modify the mouse genome How would it integrate into the genome How would you detect its integration Exon1 Intron1 Exon2 Intron2 Exon3 DNA construct NeoR gene TKHSV gene Homologous to sequences just outside of Exon2 The DNA construct contains two regions that are
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