PSet 6_7 Fall 2023 (1)

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0470

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Biology

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Dec 6, 2023

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Banner ID:______________________________ BIOL 0470 PROBLEM SET #/6/7 Fall 2023 DUE THURSDAY 11/2 BY 10:00 AM EDT Submit Via Gradescope Problem 1 (20 points) miRNAs in a new yeast model species Topics : miRNAs; yeast genetics; northern blots; western blots Learning Goals : Apply your understanding of miRNAs to a new system. Use yeast genetics and northern blots to provide insight into post-transcriptional gene regulation. Problem Setup: You are working with an interesting yeast species that has a fully functional miRNA gene regulatory pathway. This is exciting because Saccharomyces cerevisiae lacks miRNAs and now you can use this other yeast species to apply all the genetic tricks available for Saccharomyces cerevisiae to the study of miRNA mechanisms. This new yeast model system has all of the same genetic characteristics as S. cerevisiae. To study the miRNA mechanism in this species, you engineer two haploid strains: 1) One strain expressing the green fluorescent protein ( GFP ). To create this strain, you insert a gene encoding GFP into the haploid yeast genome. You don’t know where the GFP gene is inserted, but you know it has integrated into a chromosome. 2) One strain carrying a miRNA gene that produces a 21-nucleotide mature miRNA. The miRNA is perfectly complementary to a region toward the 3' end of the GFP coding sequence. (A, 4 points) The hybrid diploid you produce by fusion of the two haploid strains described above fails to express GFP even though you can easily detect GFP in the parental GFP strain. Provide a simple explanation for this result in light of the miRNA model for gene regulation. Include a complete description of the genotype of this diploid strain with respect to the two genes you've engineered (no need to come up with notation for the genotype; just describe it). [2-4 sentences]

Banner ID:______________________________ You sporulate this diploid and analyze 1000 (haploid) tetrads under the fluorescence microscope. You find three expected classes of tetrads and the frequencies of the classes suggest that your GFP gene and your miRNA gene integrated onto homologous chromosomes when you produced each haploid strain (see data table below). (B, 4 points) Draw a simple diagram representing this key chromosome when the four chromatids (2 from GFP parent, 2 from miRNA parent) are aligned during Meiosis I in the diploid strain. Mark your GFP locus with G and your miRNA locus with M. Place X(s) on your diagram to indicate the position(s) of meiotic crossover event(s) required to produce a non-parental ditype tetrad (tetrad containing two different genotypes, both are recombinant) . (C, 6 points) Complete the table below by filling in the color of each spore in the following tetrads (green means GFP is detected, beige means GFP is not detected). Label each tetrad type you observe with its “Perkins name” (T, PD, NPD). PD = Parental Ditype (tetrad containing only the two different parental genotypes) T = Tetratype (tetrad containing 4 different genotypes - the two parental and two recombinant genotypes) NPD= Non-Parental Ditype (tetrad containing two different genotypes, both are recombinant)

Banner ID:______________________________ Spore * Spore Color (either green or beige) Tetrad type (PD, T, or NPD) 950 of the following type of tetrads were observed out of 1000 total 1 2 3 4 10 of the following type of tetrads were observed out of 1000 total 1 2 3 4 40 of the following type of tetrads were observed out of 1000 total 1 2 3 4 *tetrads comprise 4 spores To study how GFP expression is regulated by the miRNA, you focus on the tetratype tetrads. You choose one tetrad and grow independent cultures from each individual spore. You isolate RNA and protein from each culture for analysis by RNA gel blot (Northern) and immuno-blot (Western).

Banner ID:______________________________ (D, 6 points) Your RNA gel blot analysis gives you strong evidence that the miRNA directs endonucleolytic cleavage (slicing) of the GFP mRNA in the region where the miRNA is complementary to the GFP coding sequence. Interestingly, the mRNA cleavage products are stable and detected on the RNA gel blot. You have to run two different blots to detect the mRNA and miRNA because their size difference is too great to detect both in one gel system. Only the mature miRNA is detectable on the miRNA blot. The GFP mRNA is 750 base pairs long (the coding sequence is 714 base pairs). The miRNA target site is 200 nucleotides from the 5' end of the mRNA. Draw in the band(s) you would expect to observe on the autoradiogram of your RNA gel blots, and label their lengths (in nucleotides):

Banner ID:______________________________ Problem 2 (20 points) The Lac Operon Topic: lac operon - analysis of cis -regulatory elements and trans factors Learning Goals: Students should be able to predict how the lac operon will behave when key components are mutated. Problem Setup: This is a map of the E. coli lac operon: ● E. coli are grown on petri dishes containing a defined medium. ● Glucose must be absent for the Promoter (P) to recruit RNA Polymerase. ● The operon is induced by lactose, or by IPTG (Isopropyl-β-D-thiogalactoside), which is not hydrolyzed by b-galactosidase (the product of the lac Z gene). ● lac I encodes the lac repressor protein, which binds lac O unless IPTG or Lactose are provided in the growth medium. ● E. coli is haploid - it has one copy of the lac operon (above) on its chromosome. This is the source of the endogenous alleles in the table below. ● F’ plasmids were used to introduce a second allele to test for complementation or to determine whether an element/gene functioned in cis or in trans. These are called F’ donated alleles in the table below. Fill in the missing information in the following chart (empty boxes). Each part (1-4) represents a different experiment. There are two different conditions for each experiment (a control and an experiment). Your goal is to fill in the table with experimental conditions and observations that unambiguously lead to the indicated conclusion. The endogenous alleles are indicated. Those donated by F’ plasmids are either given or they are left blank and need to be filled in. The presence or absence of glucose and IPTG in the media is indicated (yes/no). Whether expression of active Z or Y protein is detected is also given (yes = the enzyme activity is detected; no = the enzyme activity was not detected). The following alleles were used (use only these alleles): + = wild type for the gene/element - = a loss of function allele of the indicated gene/element. s = ‘super repressor’, disrupts ability of I gene product (lac repressor) to bind IPTG, but does not affect its ability to bind lac O.

Banner ID:______________________________ Strategy : Refer back to the examples from lecture that relate to the lac operon. Make sure you have a solid understanding of this system before attempting the problem. If you are unsure how to begin, start by playing around with the different alleles in each experiment. Do not make things more complicated than necessary!

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