MBMB451A Section1 Fall 2008 KEY These questions may have more than one correct answer 1. In a double stranded molecule of DNA, the ratio of purines : pyrimidines is (a) variable (b) determined by the base sequence in RNA (c) genetically determined (d) always 1:1 (e) determined by the number of purines in the sense strand of the DNA 2. Melting of the DNA duplex disrupts hydrogen bonds between complementary bases and hence changes their absorption characteristics. The DNA melting temperature: a. is approximately 45ºC for mammalian DNA therefore fever above 42ºC is extremely dangerous. b. Depends on the A/T content because the more A/T is present the less energy is needed to separate two strands. c. Can be defined as the midpoint of the temperature range over which the DNA strands separate. d. Can be determined by the change in the absorption at 260nm which is characteristics for the bases. e. Is the temperature at which single-strand breakage occurs (phosphodiester bonds break). 3. Denaturation of DNA : (3points) a. can involve the melting of the duplex b. can be caused by lowering of the temperature c. is reversible d. is the breakage of phosphodiester bonds e. can involve the disruption of hydrogen bonds 4. The tertiary structure of a protein refers to the: a. Presence of alpha-helices or beta-sheets. b. The sequence of amino acids. c. The unique three dimensional folding of the molecule. d. Interactions of a protein with other sub-units or enzymes. 5. Which of the following lists, correctly labels the diagram of the amino acid a. (1) carboxylic group, (2) R group, (3) alpha carbon, (4) amino group. b. (1) R group, (2) alpha carbon, (3) carboxylic group, (4) amino group. c. (1) amino group, (2) alpha carbon, (3) R group, (4) carboxylic group 1
d. (1) carboxylic group, (2) alpha carbon, (3) R group, (4) amino group. 6. Which amino acid is represented in above question: a. Glutamine b. Alanine c. Lysine d. Proline 7. Which of the following statements concerning the structure of proteins is not true? a. Side chains of amino acids can be hydrophilic or hydrophobic. b. The primary structure is the sequence of amino acids c. Proteins made of two or more polypeptide chains have tertiary structure. d. Alpha-helices and beta-sheets are examples of secondary structure. 8. Which arrow points towards the peptide bond. a. 2 b. 4 and 5 c. 1 d. 2 and 3 9. Identify the regions A and B of protein in the image a. Alpha helix (A) and beta sheets (B) b. Beta sheets (A) and alpha helix (A) c. Random coil (B) and beta sheets (A) d. Beta sheets (A) and alpha helix (B) A B 10. Two different proteins of similar molecular weight having different isoelectric point can be separated by (2point) a. Gel filtration chromatography b. Cation and anion exchange column c. Pulse field gel electrophoresis d. SDS-PAGE e. 2-dimensional gel electrophoresis 11. Which tool of recombinant DNA technology is incorrectly paired with its use: 2
a. restriction enzyme: production of RFLPs (restriction fragment length polymorphism) b. DNA ligase: enzymes that cut DNA, creating sticky ends c. DNA polymerase: used in PCR to amplify sections of DNA d. Reverse transcriptase: production of cdna from mrna e. electrophoresis: DNA sequencing 12. A researcher recovered a bit of tissue from the preserved skin of a dinosaur. The researcher would like to increase the amount of DNA available for testing purposes. He would use: a. RFLP (restriction fragment length polymorphism) analysis b. PCR c. electroporation d. Gel electrophoresis e. Southern hybridization 13. Components of DNA are a. Phosphate, ribose, pyridines, and purines b. Phosphate, ribulose, and nucleotides c. Sulfate, ribose, pyrimidines, and purines d. Phosphate and nucleotides e. Phosphate, deoxyribose, pyrimidines, and purines 14. The DNA double helix is stabilized by a. Ionic bonds b. Covalent bonds c. Hydrogen bonds d. Hydrophobic interactions e. Electrostatic interactions 15. What two enzymes are needed to produce recombinant DNA? a. endonuclease, transcriptase b. DNA polymerase, topoisomerase c. restriction enzyme, ligase d. polymerase, ligase e. transcriptase, ligase 16. What are the essential characteristics of a cloning vector a. Bacterial cells cannot survive without it. b. Bacterial cells replicate it. c. Bacterial cells take it up. d. Both B and C are correct. e. A, B, and C are correct. 17. Answer the following questions based on the structure: A B C 3
a. What does A, B and C boxes represent? Triphosphate, ribose sugar and guanine b. Write the complete name of the molecule. c. Guanosine triphosphate d. Where will the next nucleotide form the bond and what is the name of the bond. Two phosphate will be removed and next phosphodiester bond will be formed e. This kind of molecule is found in which kind of nucleic acid. Explain. RNA because it has OH at 2 and 3. 18. There are two methods of nucleotide sequencing, one is Maxam-Gilbert method and the other is Sanger s method. The advantage of the later method is a. the differential interaction of the bases with particular dye b. extension of the synthetic primer and reliable termination of DNA repair synthesis. c. The correlation of restriction sites with the end label of the DNA. d. The ability to sequence both strands simultaneously. e. That only DNA and not RNA will be identified. This allows investigation of less pure preparations and so reduces costs. 19. Which is not a step of the Southern blotting procedure. a. digestion of DNA with restriction enzyme b. ligation of DNA on to the vector c. separation of DNA fragments on gel d. transfer of the DNA fragments to a nitrocellulose membrane e. hybridization of the membrane with a label probe. 20. The polypeptide backbone conformation can be specified by its torsion angle of each amino acid residues by Φ and Ψ angles. These angles are between a. C-C and C-N b. N-C and N-N c. Cα-N and Cα-C d. Cα-C and Cα-N e. Cα-N and C-C 21. Topoisomerase IA and IB can a. relax negative supercoiling only b. relax positive supercoiling only c. IA can relax negative and IB can relax positive supercoiling d. IA can relax positive and IB can relax negative supercoiling e. IA can relax negative and IB can relax both kinds of supercoiling 22. Describe how to do DNAse I footprinting and give an example of it might be used. (8points) 4
Key words DnaseI is a nuclease, single cutting such that ladder is obtained in naked DNA control, radio label DNA at 5 end, protein bound to the DNA, protection of DNA, absence of cut fragments in a sequenceing gel. 23. Describe how proteins can recognize and bind to specific DNA sequences. Make sure to highlight the features of the DNA as well as the protein that are important in this intermolecular interaction. (7points) Keywords DNA sequence recognition ( donor and acceptor sites in neucleotide base and proteins), alpha helix used to bind inside of the major groove and minor groove recognition, amino acid of DNA binding motif, comment about helix turn helix motif. 24. What are the important components of an over-expression vector? (7 points) Selectable marker Able to replicate in bacteria High copy number Strong promoter like t7 Inducible expression Proper transcription terminator Polylinker or MCS Tag for protein purification 25. What is the propensity of different amino acids to be found on the surface or in the internal portion of proteins? (6 points) Non-polar or hydrophobic amino acids interior of the protein (Val, Leu, Ile, Met and Phe). Charged polar or hydrophilic amino acids on the surface of the protein ( Arg, His, Lys, and Asp). Uncharged polar residues primarily on the surface but can also be found in the interior of the proteins (Ser, Thr, Asn, Gln, Tyr and Thr). 26. The SDS-polyacrylamide gel electrophoresis of a protein yields two bands corresponding to molecular masses of 10 and 17 kda. After cross-linking this protein with dimethylsuberimidate under sufficient dilution to eliminate intermolecular crosslinking, SDS-polyacrylamide gel electrophoresis of the product yields 12 bands with molecular masses 10, 17, 20, 27, 20, 37, 40, 47, 54, 57 64, and 74 kda. Assuming that dimethylsuberimidate can cross-link only contacting subunits, diagram the quaternary structure of the protein. (6point) 5
74KDa tetramer of 10 and dimer of 17 27. In what order will the following proteins be eluted from a CM-cellulose ion exchange column by an increasing salt gradient at ph 7.0 : fibrinogen, hemoglobin, lysozyme, pepsin, and ribonuclease? (see reference table on last page) (4point) CM-Cellulose is cation exchange resing pepsin fibrinogen Hemoglobin ribonuclease A, and lysozyme. ph>pi ve charge ph<pi +ve charge 27. What would be the relative arrangement of the following proteins after they had been subjected to isoelectric focusing: insulin, cytochrome c, histone, myoglobin, and ribonuclease A? Sketch the appearance of a two-dimensional gel of cytochrome, myoglobin, and ribonuclease A on the next page. (6point) Insulin, cytochrome C, histone, myoglobin and ribonucleasea. Table 1 Protein Isoelectric ph Molecular Mass (kda) Pepsin <1.0 Ovalbumin (hen) 4.6 Serum albumin 4.9 (human) Tropomyosin 5.1 Insulin (bovine) 5.4 Fibrinogen (human) 5.8 g-globulin 6.6 Collagen 6.6 Myoglobin (horse) 7.0 16.9 Hemoglobin (human) 7.1 Ribonuclease A 7.8 12.6 (bovine) Cytochrome c (horse) 10.6 13.4 Histone (bovine) 10.8 Lysozyme (hen) 11.0 Salmine (salmon) 12.1 6
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