2005 7.03 Problem Set 5Due before 5 PM on WEDNESDAY, November 16, 2005.Turn answers in to the box outside of 68-120.PLEASE WRITE YOUR ANSWERS ON THIS PRINTOUT.1. You are studying the regulation of a yeast gene (His1), which is necessary for synthesis ofthe amino acid histidine. To begin your analysis of the regulation of His1, you fuse the cisregulatory region (“PHis1”) that lies upstream of the His1 open reading frame to the LacZcoding sequence. You then place this hybrid gene on a yeast plasmid. This reporter geneconstruct behaves the way you expected based on the pathway for the regulation of His1,which is as follows: his2 his3 his1Keep in mind that this model is a genetic pathway that should not be interpreted as amolecular model.You monitor the expression of the his1 gene using your reporter gene construct in order toperform a genetic screen looking for mutants that do not properly regulate expression ofhis1. In your screen, you isolate a series of haploid mutant strains that either showconstitutive or uninducible expression of his1. You identify the genes that are mutated in themutants you find, and discover that you have identified new alleles of two genes, his2 (whichlies on chromosome #1), and his3 (which lies on chromosome #5).In your screen, you isolate five strains, each of which contains one of the followingsingle mutations:his2a, which is in the coding region of his2. This mutation gives a recessivephenotype.his2b, which is in the coding region of his2. This mutation gives a constitutivephenotype.his3c, which is in the coding region of his3. This mutation gives a constitutivephenotype.his3d, which is in the coding region of his3. This mutation gives a recessivephenotype.RHis2–, which is a deletion in the cis regulatory region in front of his2.PHis1RHis2PHis3histidine2(a) Is his2a cis-acting or trans-acting with respect to his1?(b) Is RHis2 cis-acting or trans-acting with respect to his1?(c) What is the phenotype of a his2a his3d double mutant with respect to expression of his1?(d) Would the his3c mutation give a dominant or recessive phenotype with respect toexpression of his1?(e) What type(s) of mutation might his2b be with respect to his1? (Your choices are:repressor–, activator–, promoter–, UAS–, URS–, dominant negative repressor, dominantnegative activator, super-repressor, super-activator.)(f) You cross a his3d haploid mutant strain to a his2a haploid mutant strain. What is thephenotype of the resulting diploid with respect to expression of his1?(g) You induce sporulation of the diploid from part (f). You analyze 90 tetrads. Three distincttetrad types are obtained. Below, fill in each blank with the phenotype of each of the sporesthat is not provided to you.Type 1: Type 2: Type 3:regulated (wt) ___________________ constitutive__________________ ___________________ ____________________________________ ___________________ __________________constitutive ___________________ __________________(h) How many “Type 3” tetrads would you have most likely observed?32. You are studying the metabolism of a sugar called struliose by yeast cells. (Note thatyeast will use struliose even when glucose is present.) You have already isolated one genethat is necessary for the use of struliose as a carbon source. This gene is inducedwhenever struliose is present. You want to do a genetic procedure (i.e. a screen orselection) to look for more genes involved in struliose metabolism, and you have tworeagents that could help you do this. One reagent is a reporter gene that you have created byattaching the promoter region of the known struliose-utilization gene to the open readingframe for E.coli lacZ. The other reagent is a form of struliose (called toxo-struliose) that canbe metabolized in the same way as struliose, but when it is metabolized, it creates abyproduct that is toxic to yeast cells. You have a collection of thousands of haploid yeast,and each yeast is mutant in a different gene. However, you don’t know which of these yeastare mutant in “struliose metabolism” genes (versus which yeast are mutant in any of theother genes in the yeast genome that have nothing to do with struliose metabolism).(a) Outline a genetic procedure that you would do to find more genes involved in struliosemetabolism. In your procedure, use the reporter gene (but not toxo-struliose). To outlineyour procedure, include: i) the type(s) of growth medium you would plate your yeastmutants on (i.e. what would have to be added to a basic growth medium that containseverything necessary for yeast to grow except a carbon source), ii) how you would identifythe yeast mutants you are looking for (i.e. what would mutants and non-mutants look like oneach type of growth medium), and iii) whether this method is a screen or a selection.i)ii)iii)(b) Outline a genetic procedure that you would do to find more genes involved in struliosemetabolism. In your procedure, use toxo-struliose (but not the reporter gene).i)ii)iii)43. You have a true-breeding mouse that displays the phenotype of big feet. This phenotypeis caused by a specific allele of the “FT1” gene called FT1*. You isolate the FT1 gene fromthis mutant mouse, and inject it into a fertilized egg produced by the mating of two wild-typemice. You then transfer this injected fertilized egg into a pseudopregnant mouse. Themouse that is born has big feet.(a) What specific conclusion can you draw regarding FT1* from this experiment?(b) Which breeding experiment could you have done to reach the same conclusion that youreached from part (a)?You make a transgenic mouse that is transgenic for a gene that is involved in determiningpetal color in petunias. This mouse has no detectable mutant phenotype. You then matetwo transgenic mice together to generate a mouse that has two copies of the sametransgene. These TG+/TG+ mice now have a phenotype of slow movement. Youhypothesize that this slow movement is caused either:-- by the presence of two copies of the petunia transgene (for unknown reasons)-- because each of the transgenes disrupted one copy of the “Dext” gene, a gene that isimportant for mouse motor skillsThe scenario in this question asks a biological question that can be addressed bycreating genetically engineered mice. When creating engineered mice, the following8 steps need to be
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