Problem set questions from Exam 3 Eukaryotic Gene Regulation Genome Modifications in Eukaryotes Population Genetics Characterizing novel pathways that control the expression of yeast genes 1 You are studying regulation of the yeast enzyme glutamine synthetase GS which is encoded by the GLN1 gene You have isolated two mutants designated gln2 and gln3 that give decreased GS activity Mating of either gln2 or gln3 haploids to wildtype haploids produce heterozygous diploids that show normal amounts of GS expression When you cross either a gln2 or gln3 haploid strain to a gln1 haploid strain the resulting diploids show normal expression of GS a From these experiments classify the gln2 and gln3 mutations in terms of their basic genetic properties dominant vs recessive cis vs trans uninducible vs constitutive and explain the rationale behind your conclusions Based on these properties make a proposal for the nature of the wild type regulatory functions of the GLN2 and GLN3 genes b Diagram two different linear models and one parallel model that could illustrate how the GLN1 gene is regulated by the wild type GLN2 and GLN3 genes The GLN1 gene shows a rather complex regulation in response to two different amino acids When either glutamate glu or glutamine gln is added to the medium the amount of GS expression diminishes when both glutamate and glutamine are added to the medium GS expression is shut off completely The effects of different mutants on the response to glu and gln are shown below Units of GS activity glu gln glu gln wild type 100 50 50 0 gln1 0 0 0 0 gln2 50 50 0 0 gln3 50 0 50 0 c Which of the models from part b best fits these experimental results Diagram a complete model for the regulation of GLN1 that includes the effects of glu gln wild type GLN2 and wild type GLN3 d Based on your model for part c how would you expect a gln2 gln3 double mutant to behave Next you decide to evaluate the cis regulatory DNA sequences found in front of the GLN1 open reading frame To do this you first fuse these regulatory DNA sequences to the LacZ coding sequence and then place this hybrid gene on an appropriate yeast plasmid You find that cells carrying this reporter gene express LacZ activity under the same conditions that GS is expressed in wild type cells This tells you that the cis regulatory region you have selected contains all of the necessary cis acting sequences for normal regulation The figure below shows the effect of six different 50 basepairlong deletions in the cis regulatory region on the amount of galactosidase activity expressed by the reporter gene wt 300 250 200 150 100 50 1 LacZ 2 LacZ LacZ 3 1 LacZ 4 LacZ 5 6 LacZ LacZ Units of galactosidase glu gln 100 50 50 50 50 0 100 50 50 50 0 50 50 0 50 100 50 50 0 0 0 e Describe the cis acting elements in the GLN1 cis regulatory region that are evident from these experiments giving both their positions and as much of their wild type function as you can deduce f How many units of galactiosidase would you expect to be expressed in gln2 mutant yeast from a reporter gene construct carrying deletion 1 with neither amino acid added with only glu added with only gln added g How many units of galactiosidase would you expect to be expressed in gln2 mutant yeast from a reporter gene construct carrying deletion 4 with neither amino acid added with only glu added with only gln added 2 Consider a eukaryotic gene regulatory pathway where a small molecule X activates the expression of a reporter gene You have isolated loss of function mutations in two different genes A and B both of which give uninducible expression of the reporter Genes A and B are not linked to each other and neither gene is linked to the reporter a Assuming that the regulatory factors encoded by A and B act in series there are two possible orders in which these two regulatory factors can act Draw out these two models showing the relationships between the wild type regulatory functions of A and B and the reporter Also be sure to indicate where and how the inducer X acts b In order to distinguish between the two models from part a an epistasis test would be useful Because the mutations that have been isolated in the A and B genes have the same phenotype uninducible it is not possible to perform an epistasis test using these alleles Fortunately you are able to isolate an allele of gene A that gives constitutive expression of the reporter This allele called As causes a dominant phenotype of constitutive reporter expression Describe in molecular terms how the allele As affect the normal regulatory function of A given each of your models from part a c Assume that you are studying this regulatory pathway in yeast and you wish to perform an epistasis test by constructing the As B double mutant To do this you cross a MATa As B haploid strain to a MAT A B haploid strain and induce sporulation of the resulting diploid You examine the resulting tetrads For each of the two models from part a give the types of tetrads that you would expect and their relative frequencies The tetrad types should be described by the phenotypes constitutive uninducible or regulated of the four spores in each tetrad d Now assume that you are studying this regulatory pathway in Drosophila To perform the epistasis test you cross a As As B B male to a A A B B female For each of the two models from part a give the expected phenotype of the F1 flies from this cross Now you cross the F1 flies among themselves to produce F2 flies For each of the two models give the expected ratio of constitutive uninducible or regulated phenotypes among the F2 flies 3 You are studying the regulation of an enzyme in yeast To begin your analysis of this regulation you first fuse the cis regulatory region found upstream of the coding region of the gene encoding this enzyme to the LacZ coding sequence You then place this hybrid gene on an appropriate yeast plasmid You are relieved to find that cells carrying the hybrid gene do not express galactosidase activity unless the known inducer for the enzyme synthesis is present meaning that the regulatory region you have selected contains all of the necessary cis acting sequences for normal regulation You next identify two different mutants that show abnormal regulation of your reporter gene construct Mut1 gives constitutive expression whereas Mut2 shows uninducible expression a Are Mut1 or Mut2 cis or trans acting Explain Next you cross a Mut1 haploid yeast strain to a Mut2 haploid yeast strain
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