12 Jun BIO 201 Nuclear and cytoplasmic fractions were purified
1) Nuclear and cytoplasmic fractions were purified from eukaryotic cells and RNA was purified from each fraction. A careless student labeled the RNA samples, #1 and #2, but forgot which one was extracted from the cytoplasmic fraction. To determine the origin of samples #1 and #2, you decide to perform the following experiment: Each sample is submitted to gel electrophoresis to resolve RNA molecules based on their size. After electrophoresis, the RNA content of the gel is transferred onto a membrane. The membrane is probed with radiolabeled oligonucleotides: ACAACCACAC and then placed into contact with an X-ray film to determine the position of the RNA molecules recognized by the radioactive probe. A similar experiment is conducted using a different radioactive probe whose sequence is TTTATT.
The developed X-ray films are shown in the following figure.
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a) During polyadenylation, what is the function of the pre-mRNA sequence corresponding to “GTGTGGTTGT” on the non-template DNA strand?
b) Identify the sample (#1 or #2) extracted from the cytoplasmic fraction. Give two reasons to explain your answer. 2) mRNA was purified from wild type yeast (WT) and from two mutant strains of yeast (M1 and M2). Note that yeast is a unicellular eukaryote. A series of experiments were performed with these 3 types of yeast cells.
Experiment 1: b-galactosidase (b-gal) mRNA was purified, submitted to gel electrophoresis and transferred onto membrane as described above. The membrane was probed with either a radioactive oligonucleotide that recognized a sequence unique to b-gal mRNA or radioactive oligo (dT) (Sequence TTTTTTTTTT). Note that the oligo(dT)- target sequence hybrid is stable if the target sequence contains more than 6 adjacent As.
The developed X-ray films are shown in the following figure.
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Experiment 2: The protein b-galactosidase is an enzyme that converts X-Gal, a colorless substrate, into a blue product. mRNA extracted from slices of the electrophoresis gel corresponding to the regions of the film labeled a and b (see above) was used for in vitro synthesis of b-galactosidase. The amount of b-galactosidase synthesized was measured by adding X-gal to the reaction and by measuring the amount of blue product formed. This experiment shows that identical quantities of mRNA extracted from a and b produced identical quantities of b-galactosidase.
a) Explain the size heterogeneity of the b-gal mRNA in the wild type strain. Note: the result from experiment 2 should give you a clue.
b) The genes mutated in strain M1 and M2 are different. In both mutant strains the gene mutation results in the production of an inactive protein. Propose a function for the proteins inactivated in M1 and M2.
3) The gene coding for b-galactosidase is under the control of an inducible promoter. Therefore, the gene is transcribed only when a diffusible inducer is added to the yeast culture.
Experiment 3: The inducer was added to the yeast culture for 2 minutes, and then the cells were harvested and resuspended into a fresh culture medium containing a transcriptional repressor of the b-galactosidase gene. Samples of yeast cells were taken from the culture at different time intervals before or after the addition of the repressor. The b-galactosidase activity in these samples was immediately measured. Alternatively, mRNA was extracted immediately after the addition of the repressor and stored on ice. At different time intervals, these mRNA samples were translated and the b-galactosidase activity in these in vitro translation reactions was measured.
a) Is experiment 3 a pulse-chase experiment? Briefly explain
b) The following graphs represent the results of experiment 3.
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Describe the two biological properties of the polyA tail illustrated by experiment 3. Briefly explain.
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