MCB 102 University of California, Berkeley August 11 13, Problem Set 8

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1 MCB 102 University of California, Berkeley August 11 13, 2009 Isabelle Philipp Handout Problem Set 8 The answer key will be posted by Tuesday August 11. Try to solve the problem sets always first without the answer key. Remember, your goal is to be able to solve biochemical problems in the exams. If you can not solve one of the problems try to solve it together with one of your classmates or ask your GSI during discussion section. Don t just wait for the answer key. The answer key should only confirm that you solved the problem correctly! Exercises and Problem Sets are for extra practice and exam preparation, and should not be turned in. Recommended problems from the book: Chapter 28: 1, 2, 4, 5, 6, 7 Chapter 9: 1, 2, 4, 5, 6, 7 1. The Genetic Code (a) Are all amino acids encoded by the same number of codons? (b) Which amino acid(s) are encoded by the most codons? (c) Which are encoded by only one codon? (d) Which are encoded by only one codon? (e) What are the trna anti codons that can bind to the tyrosine codons? Note the 5 and 3 ends. (f) What is special about the amino acid methionine? 2. Regulation of Gene Expression Mutations can alter the function of an operon. Predict how the following mutations would affect the Lac Operon gene transcription (i) in the presence of lactose and (ii) in the absence of lactose? (a) Mutation of the Operator so that the repressor can no longer bind to it. (b) Mutation of the Promoter so that it is no longer seen by RNA polymerase. (c) Mutation of the Promoter for the laci repressor so that it is no longer bound by RNA polymerase.

2 2 Handout : Problem Set 8 3. Gene expression You want to express a human keratin protein, so you need to integrate the human gene into a bacterial plasmid. If you cut out the human keratin gene directly from the human DNA genome and insert it into the plasmid, will the bacteria be able to express this gene for you? If not, why not? What additional steps would you need to do to get the bacteria to express your gene? 4. The Genetic Code II In studies of the evolution of amino acid sequences in Drosophila, the following changes have been observed. Using the genetic code in your text, determine a set of triplet codes in which only a single nucleotide change produces each amino acid change. (Each arrow indicates one amino acid change caused by a single nucleotide change from the previous codon).

3 Handout : Problem Set Gene Analysis Below is a picture of human genomic DNA that contains a gene found to be abnormally regulated in skin cancer cells. There is a ruler below the genomic DNA that indicates every 100 base-pairs (the entire region shown is 1800 bp long). You want to study the gene further to learn about its function, and whether mutations in the gene are found in skin cancer patients. You identified four sequences that can be cut by the Restriction enzymes HindIII, AvaII and SmaI, as marked above. (Thursday and Tuesday) (a) If you cut with SmaI, how many fragments do you expect to get? (b) You separate the fragments based on size using gel electrophoresis. Draw a picture of the gel with what your fragments would look like on the gel. Label the approximate sizes of the fragments. Add in the + and poles of the gel. (c) If you cut with HindIII, how many fragments will you get? (d) If you cut with HindIII and SmaI how many fragments will you get? (e) Lets say you wanted to amplify the promoter region using PCR to identify transcription factor binding sites. Draw on the picture above to show where you would have your PCR primers placed to specifically amplify this region. Use arrows ( ) to represent your primers and their required 5 3 orientation in the picture above. (f) Why is denaturation the first step during PCR? 6. Trp operon What would be the effect of a deletion of region 4 of trpl on regulation of transcription. 7. PCR Why is a heat-stable DNA polymerase used for the polymerase chain reaction (PCR)?

4 4 Handout : Problem Set 8 Exam Questions from Operon You are an undergrad research assistant in a UC Berkeley lab and are working in bacteria on the metabolism of the monosaccharide, theose. Metabolism of theose requires the enzymes X, Y, and Z. The genes encoding these enzymes are part of one operon, and the product of gene N (N protein) regulates the transcription of these three genes. In a normal cell, protein A is always produced. A diagram of this operon is shown below. (a) Give a brief definition of an operon. (b) In a cell where there is a high level of N protein, you detect no transcription of genes X, Y, and Z. You conclude that... The N protein is (circle one) a repressor. an activator. (c) In further study, you discover that transcription of the N gene is controlled by protein A (protein A is the product of gene A shown in the diagram), and theose binds to the N protein. You examine the transcription of genes N, X, Y, and Z in cells where gene A is normal (A+) and where gene A is not functional (A-) and in the presence (+) and absence (-) of theose. The data is shown below. i. Given the results above, what does protein A do? ii. Given the results above, what does theose do?

5 Handout : Problem Set Restriction digest You just isolated a novel recombinant clone and purified the desired insert (a 10,000 bp long linear duplex DNA) from the vector. Now you wish to map the recognition sequences for restriction endonucleases A and B. You cleave the DNA with these enzymes and fractionate the digestion products according to size by agarose gel elecrophoresis. In lane 1 you cut only with A, in 2 with B and in 3 with A+B. Comparison of the pattern of DNA fragments with DNAs of known sizes yields to the following result: Draw a restriction map of the insert showing the relative positions of the cleavage sites to one another: