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exm3_ans - MIT Department of Biology 7.28 Spring 2005...

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______________________________ MIT Department of Biology 7.28, Spring 2005 - Molecular Biology 7.28 Spring 2005 Exam Three Name ______________________________ Question 1 _____/30 points Question 2 _____/20 points Question 3 _____/21 points Question 4 _____/29 points Total _____/100 points 1
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7.28 Spring 2005 Exam Three Name ______________________________ Question 1 (30 points total). You are studying the genetic control of muscle formation during mouse development. Comparisons of heart muscle vs skeletal muscle revealed several differences in key structural proteins. For example one protein, called p78 (with an apparent molecular weight of 78kDa) in skeletal muscle appears to be absent in heart muscle, and replaced with a slightly larger protein, (called p80). To investigate the source of these differences in protein expression, you request a cDNA clone for p78 from another lab. Your first hypothesis is that p78 and p80 are encoded by alternatively spliced mRNAs from the same gene. 1A (6 points ). Describe a simple experiment that could provide support for or against this model (no sequencing needed). You have available both heart and skeletal muscle cells in sufficient quantity to isolate RNA and/or DNA. Describe a result from this experiment that would strongly argue against this alternative splicing model. Do a Northern blot: Isolate mRNA from both cell types using an oligo dT column(use cytoplasmic fractions to get mRNA). Use the p78 cDNA to make a radiolabeled probe. Run mRNA on a gel, transfer, and probe. If p78 and p80 are alternatively spliced mRNAs from the same gene, you will see a band for both cell types, most likely of different sizes. A result that would argue strongly against alternative splicing is if the probe does not recognize anything in the heart muscle cell lane, suggesting that these two proteins are encoded by different genes. 1B (4 points ). Below is a Northern blot. The RNA samples are from skeletal muscle cells and the p78 cDNA has been labeled and used as a probe. The two samples are: nuclear RNA (N), and cytoplasmic RNA (C). Based on these data, draw a schematic of the pre-mRNA that encodes p78. Label the length of segments to the best of your ability. 5’CAP--UTR+exon1(650 nt)—intron(1000 nt)—exon2+UTR(250 nt)–3’polyA tail 2
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7.28 Spring 2005 Exam Three Name ______________________________ Size Markers 1 2 RNA sample: 1900 1250 1000 800 ntds Film: p78 cDNA probe 650 N C 1C (4 points). Which of the RNA molecule(s) on this Northern blot would you expect to interact with the splicing factor U2-AF? Circle the band(s) on the figure, and briefly explain your answer. U2-AF binds the Py tract and 3’SS. It will bind the band at 1900 nt because this represents the unspliced pre-mRNA. It may bind the band at 1250 nt because this is the intronic lariat + exon2. However, U2-AF normally is off after the first trans-esterification reaction and the structure of the RNA has changed. U2-AF may not be able to bind this structure even in an in vitro binding experiment.
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