课程名称︰核酸化学与遗传机制 课程性质︰系内选修 课程教师︰谢道时 开课学院：理学院 开课系所︰化学系 考试日期（年月日）︰2011/11/8 考试时限（分钟）： 60 mins 是否需发放奖励金：是 （如未明确表示，则不予发放） 试题 : (36) 1. Nucleic acids are made of base, sugar and phosphate. The following structures show the examples of bases (A and B), and sugar (C). NH2　　　　　　 　 　NH2 OH │ │ │ C N C CH2 O OH // \ ／ ＼ // \ │／ ＼│ N C CH N CH │＼＿／│ │ ∥ ／ │ ∥ H ││ H HC C─NH C CH HO OH \\ / // \ // N O N Ａ Ｂ C Name the above three. Number the key (skeleton) atoms. Remember some of them may have the prime tags. Mark the position (or the numeral of ring atom) of base in A and B that is attached to the suger. In addition to A and B, name the other two bases in DNA. Name the other two bases in RNA. The suagar shown in C is found in RNA. Name the sugar that is found in DNA, and mark the difference in C for the DNA sugar. Mark the positions in A, B or C where phosphate is attached to. The phosphate can be present as monoester or diester. Diester has__ negative charge (s) and monoester has __ negative charge(s). Bases are sometimes modified covalently. For example, A and B can be both methylated. Mark the position (or name the numeral of the ring atom) where methyl group can be attached to (in A and B). (6) 2. UV absorption spectrometry is used widely as a method to quantify and determine DNA structure. Fill in the blanks about UV spectroscopy. The Maximal absorption in near UV region occurs at the wavelength of ___ nm. During denaturation of DNA (ds to ss DNA) the absorption at above wavelength will (increase or decrease) by about ___%. This shift in absorption is due to the ____--_____ interactions in the stacked bases in ds DNA. (4) 3. One approach to probe a specific DNA sequence is through the use of molecular beacons. Briefly describe the two critical components in molecular beacons, one for sequence probe and the other for optical read-out. (6) 4. Protein crystallographers have defined the binding functions of many commonly observed structural motifs. Select the binding partner from a list of candidates (right column) for each of the following structural motifs. There may not be one-to one correspondence. __SH2, src homology 2 a. DNA __SH3, src homology 3 b. phospholipid __PH, pleckstrin homology (from phospholipase C) c. sugar __EF hand (from calmodulin) d. phosphotyrosine __Helix-turn-helix (from Cro and lambda repressor) e. Proline-rich sequen -ce __Basic-Ieucine zipper (from transcription factor AP1) f. Calcium ion (2) 5. TATA Binding Protein (TBP) has a central beta-sheet bundle that can approach DNA from the minor groove. What is the amino acid that is intercalated (inserted between basepairs) into DNA? Lysine, or Serine, or Phenylalanine, or Histidine, or Alanine (circle the right one). The intercalation of TBP can cause what structural change in DNA? Kinking, Unwinding, wrapping (Circle one). (2) 6. Beta-hairpin/ribbon in IHF(Integration Host Factor) can approach DNA from minor groove and can cause a large structural change: Kinking, Unwinding, wrapping (Circle one). (8) 7. Polymerase Chain Reactions (PCR) are commonly used for amplifying/synthesizing DNA segments bounded by a pair of oligonucleotide primers. It requires the use of a thermostable DNA polymerase. Briefly explain why. If you carry out n cycles of amplification, what is the mathematically possible fold of amplification (i.e. if you start with one copy of target sequence, how many copies of DNA fragments you will obtain.at the end of PCR reactions)? (4) 8. DNA crossovers are defined as positive or negative writhes (related to supercoiling). Crossover in isolation carries no information in signs (A), but it does if the connection is known (B and C). Use the vector rotation method to determine the writhe in B and C. ║ ╒╗ ║ ╥ └╥ ╓╥ ╚╝ (14)9.From the results obtained in the previous question, it is possible to design a machine (enzyme) to generate a unidirectional change in linking number (or writhe). The following diagram shows a proposed mechanism for a DNA topoisomerase. ┌──┐ ┌──┐ │ ┌│─┐ ┌│──│─┐ │ └┘ │ → └───┘ │ │ │ │ │ └────┘ └────┘ Determine the linking numbers in A and B, respectively. Calculate the linking change in such a reaction. An enzyme catalyzes repetitive cycles of A to B reactions will lead to what type of linking changes? What is the name of DNA topoisomerase proposed for such a reaction? Do you expect such an enzyme will require ATP hydrolysis for the cycling of A->B reaction? Why? (12) 10. DNA knotting may be associated with intracellular genome condensation and entanglement. DNA topoisomerase II has been proposed to resolve DNA knots. The following diagram illustrates the reaction with the simplest topological knots (trefoils). Identify the crossovers, mark the linking number for each one, and sum up the linkage for each molecule. Mark the site where topoisomerase acts to mediate the strand passage for reaction I and II, respectively. Are the products of reactions I and II topologically knotted? If no, what are they (in terms of DNA topological state)? ┌──┐ ┌──┐ ┌─│────┐ ┌─│──│─┐ └────┘ │ → └────┘ │ └────┘ └────┘ ┌──┐ ┌──┐ ┌────│─┐ ┌─│──│─┐ └─│──┘ │ → └─│──┘ │ └────┘ └────┘ (6) 11. Briefly describe what a G-quartet (G quadruplex) is? Does it use Watson-Crick type of hydrogen bonding? Where on each chromosome can this type of structure most likely be found? (5) 12. The so-called nucleosome paradox can be simplified as follows. From electron microscopic measurements (determining the number of nucleosomes per DNA molecule), and topological shift assays (determining the total linking number changes per DNA molecule), each nucleosome was determined to reduce the linking number by about 1.25 through left-handed wrapping of DNA. However the X-ray crystal structure suggests that the wrapping was actually 1.75 turns per nucleosome. Each set of data are precise enough so that the discrepancy can not be due to experimental error. How would you rationalize it? --

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