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The differentiation of neuronal cells is regulated by global changes at all levels of gene expression. Scientists have identified a micro-RNA called miR-A that promotes neuronal differentiation. 1) The plasmid pGFP containing a DNA sequence coding for the green fluorescent protein (GFP) under the controlled of a constitutive promoter. The 3’UTR sequence of P1 was added immediately downstream of ther GFP sequence in this plasmid. pGFP was built to test the role of miR-A. Three population of cells were prepared by co-transfection. • Population #1: cells were co-transfected with pA, a plasmid expressing miR-A, and pGFP • Population #2: cells were co-transfected with pA and a control plasmid (pREST) in which the GFP sequence is fused with a 3’UTR that does not match the miR-A sequence • Population #3: cells were co-transfected with pA and plasmid pSCP1 in which the GFP sequence is fused with a 3’UTR known to be a target for miR-A. The level of fluorescence inside co-transfected cells was measured and the data are summarized in the following table. Plasmids Fluorescence pA + pGFP 36 pA + pREST 100 pA + pSCP1 35 Is the 3’-UTR sequence of P1 targeted by miRA? Explain. 2) Differentiating neurons express a protein related to P1 called P2.

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e) When expression plasmid P is purified from a different a bacterial strain that is not E. coli, the digestion of circular P with EcoRI and BamHI produces a single 5,000 bp linear fragment. Propose an explanation for this difference. A complete sequence of the plasmid indicates that the result cannot be explain by mutations. Hint: It has something to do to the change of bacterial strain.

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Jenny Wu

Numerade educator

Your goal is to clone DNA fragments into an expression plasmid. You select an expression plasmid (P) that could be used to transcribe the sequence of the cloned DNA fragments. 1) The table below shows the results of digestions experiments on the original and circular expression plasmid (P) with combinations of restriction enzymes. You know that the multiple cloning site of P contains a unique sequence (E) cleaved by the restriction enzymes EcoRI. Circular plasmid P digested with Sizes of restriction fragments EcoRI. 5,000 bp EcoRI + PstI. 2,200 bp + 2,800 bp BamHI 500 bp + 4,500 bp EcoRI + BamHI. 500 bp + 1,800 bp + 2,700 bp BamHI + PstI 500 bp + 4,000 bp EcoRI + BamHI + PstI 500 bp + 1800 bp + 2,200 bp a) What is the length (in bp) of the expression plasmid P? Explain your reasoning. b) How many sites cleaved by PstI sites are detected in P? Explain your reasoning. c) How many sites cleaved by BamHI sites are detected in P? Explain your reasoning.

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Bacteriophage Titer (pfu/μl) Type of bacteriophage Neutral pH wash Low pH eluate M13-WT 100 5 M13-EPI 12 95 b) Based on the table above, determine which of the following statements are correct. For statement (correct or not) provide a brief explanation: A. At neutral pH, M13-WT has a high affinity for thrombin B. At neutral pH, M13-EPI has a high affinity for thrombin C. A decrease of the pH promotes the release of both M13-WT and M13-EPI from the thrombin-beads D. A decrease of the pH inhibits the release of both M13-WT and M13-EPI from the thrombin-beads E: I don’t have enough information to evaluate the effect of low pH buffer on the release of M13-WT F. I don’t have enough information to evaluate the effect of low pH buffer on the release of M13-EPI G: I have enough information to evaluate the effect of low pH buffer on the release of M13-WT H. I have enough information to evaluate the effect of low pH buffer on the release of M13-EP

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A pharmaceutical company is interested in developing an inhibitor of human thrombin, a protease involved in blood coagulation. EPI is the only known human protein that inhibits thrombin but unfortunately EPI has a low affinity and specificity for the protease. To possibly solve this problem, researchers have engineered a recombinant M13 phage that displays a modified version of the major coat protein that is fused with EPI at its N- terminus. 1) Thrombin immobilized on magnetic beads was mixed with an equal amount of two types of M13 bacteriophage: wild type, expressing normal major coat proteins (M13-WT) and recombinant, expressing major coat proteins fused with EPI (M13-EPI). After incubation, • The beads were washed with a neutral pH buffer, the wash solution was saved and its phage titer was measured using a plaque formation assay. • The washed beads were then incubated with a low pH buffer resulting in the elution of bacteriophage bound to the beads. The bacteriophage titer of the eluate was then measured. A functional thrombin inhibitor is defined by its ability to form a stable complex with immobilized thrombin. Bacteriophage Titer (pfu/μl) Type of bacteriophage Neutral pH wash. Low pH eluate M13-WT 100 5 M13-EPI 12 95 a) To test for binding specificity, a control experiment, not listed in the question, was done. Could you briefly suggest the nature and goal of this control experiment?

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Two strains of E. coli, B and K, can be infected by wild type (WT) and rapid lysis mutant (RL) T4 bacteriophages. a) A lawn of bacteria on a petri dish is infected by a mixture of WT and RL bacteriophages. Assuming a high bacteria strain B/bacteriophage mixture ratio is used for the experiment, what is (are) the size (s) of plaques (large or small) produced? In your explanation, include why RL bacteriophages produce large plaques. b) A lawn of bacteria on a petri dish is infected by a mixture of WT and RL bacteriophages. Assuming a high bacteria strain K/bacteriophage mixture ratio is used for the experiment, what is (are) the size (s) of plaques (large or small) produced? Briefly explain your answer. 2) At the end of the first step, the total concentration of recovered bacteriophages is 5 x 106 bacteriophages/ml. In the second step, 0.01 mL of the recovered bacteriophages are added to 9.99 mL of SM buffer, and 0.01 mL of this diluted bacteriophage solution is plated on a lawn of E. coli, strain B. We will assume that all the bacteriophages recovered in step 1 are infectious. a) Calculate the total number of plaques (large and small) you expect on the plate of E. coli B. Show your calculation. b) If the frequency of recombination between the 2 mutants is 5%, how many plaques would you expect when 1 mL of the diluted bacteriophage solution (see above) is plated on E. coli strain K. Show your calculations. 3) Pairwise complementation assays are done between 4 mutants RL1, 2, 3, and 4. Each mutant carry a single mutation The following table indicates the number of plaques (large or small) are obtained after plating, on E. coli strain B, the bacteriophages produced during the first step of the complementation assay. RL2 RL3 RL4 RL1 >100 > 100 <10 a) Explain why the number of plaques differ in each complementation assay. b) The experiment was performed under the same conditions, except the phages were plated on a lawn of E coli. strain K. How many plaques (few or many) would you expect for each complementation assay? c) Based on these 3 complementation assays what is the minimum number of complementation groups for this mutation? Briefly explain. d) Do mutants RL2 and RL3 belong to the same complementation group? Explain your answer

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HP1 is a DNA binding protein that interacts with a specific sequence (TGCTTATTC) found in a promoter proximal element. You want to analyze, by a Dnase I footprinting assay, the binding of HP1 and its binding partner PA on the identified promoter proximal element. In each assay, you combine a radiolabeled fragment of DNA that binds to HP1 and a specific combination of proteins. DNase I cleaves unprotected DNA sequences at random position leading to a series of DNA fragments of variable length. After digestion with DNase I, the proteins were removed from the DNA fragments and the DNA fragments were resolved by gel electrophoresis. The gel was then exposed to film to visualize the radioactive DNA fragments produced by DNaseI digestion. the results for each combination of proteins is below: Lane 1: DNA alone, no protein added in the nuclease assay. Lane 2: DNA + 5 ng (nanograms) of HP1 Lane 3: DNA + 5 ng of HP1 + 2ng of OCT1 (a protein known to interact with HP1) Lane 4: DNA + 5 ng of HP1 + 10 ng of OCT1 Lane 5: DNA + 5 ng HP1 + 5ng PA Lane 6: DNA + 5 ng PA b) The same experiment was performed with a mixture of PA, OCT1, and radioactive DNA. Which band pattern *(lanes 1, 2, 3, 4, or 5) would you expect? Briefly explain. c) Explain how DNA binding proteins can recognize a specific sequence without opening the DNA double helix.

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HP1 is a DNA binding protein that interacts with a specific sequence (TGCTTATTC) found in a promoter proximal element. 1) What is the location of a promoter proximal element and what is its function? 2) You want to analyze, by a Dnase I footprinting assay, the binding of HP1 and its binding partner PA on the identified promoter proximal element. In each assay, you combine a radiolabeled fragment of DNA that binds to HP1 and a specific combination of proteins. DNase I cleaves unprotected DNA sequences at random position leading to a series of DNA fragments of variable length. After digestion with DNase I, the proteins were removed from the DNA fragments and the DNA fragments were resolved by gel electrophoresis. The gel was then exposed to film to visualize the radioactive DNA fragments produced by DNaseI digestion. the results are below for each combination of proteins: Lane 1: DNA alone, no protein added in the nuclease assay. Lane 2: DNA + 5 ng (nanograms) of HP1 Lane 3: DNA + 5 ng of HP1 + 2ng of OCT1 (a protein known to interact with HP1) Lane 4: DNA + 5 ng of HP1 + 10 ng of OCT1 Lane 5: DNA + 5 ng HP1 + 5ng PA Lane 6: DNA + 5 ng PA a) For each lane (1 to 6) give a brief interpretation of the results. Propose how PA and OCT1 might interact with HP1. In your explanation, provide a brief interpretation of how each lane supports your proposal. More specifically: Role of PA : Role of OCT1 : Lane 1 Interpretation : Lane 2 Interpretation: Lane 3 Interpretation : Lane 4 Interpretation : Lane 5 Interpretation : Lane 6 Interpretation :

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Katlin Koehn

Numerade educator

What is the one important feature of Mendel’s selection of traits? What are the three Mendelian Laws?

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Dave Kratz

Numerade educator

dominance is a deceptively simple biological phenomenon. But it is applied in other fields. Could you give at least specific examples.

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Olivia A.