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BIOL 308
Dragana Miskovic

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1. Thinking question re DNA/RNA structure: Certain chemical agents acting on DNA could convert cytosine to uracil through the process of deamination (chopping off the amino group). This mutation is routinely repaired by the existing repair mechanism (uracil is removed and it gets replaced by cytosine). Knowing this, how would you explain why DNA contains thymine and NOT uracil. If Uracil once existed in DNA, the repair mechanism would have replaced it with a cytosine which is not complementary to Adenine, therefore the previously stable A=U will now be A C without a bond between them which is unstable and will breakdown. Evolutionarily, this is a negative force and the organisms with Uracil in DNA will be selected against. A=T on the other hand is stable and therefore, the evolutionary force will tend to favor it. 2. How does complexity of bacterial genome differ from that of eukaryotic (calf) genome? E-Coli shows a slower rate of re-naturation at the beginning, the rate then gets faster and eventually peaks and remains steady. This implies E-Coli have no repetitive sequences; the whole genome is one unique sequence. On the other hand, the calf genome starts renaturing quicker meaning there are lots of repetitive sequences. The rate then slows down as moderately repetitive sequences are renatured and finally becomes very slow as the unique sequences start renaturing (slow at first then gets faster as the complementary sequences find one another). 3. Explain C value paradox. C value is the DNA content of a haploid cell. There is no correlation between the amount of DNA (size of genome) and the apparent complexity of organisms. 4. List and briefly explain factors that influence DNA renaturation kinetics. a. Size of the DNA fragment (indirect) the larger the size, the harder it is for complementary bases to find each other. b. Complexity of genome (indirect) the more complex, the harder it is for complementary bases to find each other. c. DNA concentration (direct) complementary single strands must find each other. d. Ionic strength (direct) higher salt concentration will help neutralize –ve charge of DNA and therefore help annealing. e. Time (direct) f. Temperature. (indirect) 20-25 degrees below Tm. g. pH 5. You have found a new species of insects. To evaluate the complexity of the genome of this species, you isolate genomic DNA from, fragment the DNA to uniform 500 base pair pieces, denature the DNA and measure the rate of reassociation. Your data is represented in the curve below (sorry for the bad drawing): (a) How many classes of DNA (in respect to sequence complexity) are found in this organism? (b) What can you say about the relative complexity of each class? What fraction of the genome falls into each class? Highly repetitive Moderately repetitive Unique >1000 copies >10 copies 1-10 copies Less complex Moderately complex Very complex 25% 25% 50% 6. List three (3) differences between prokaryotic Topoisomerase I and Gyrase. Topoisomerases are enzymes that recognize and regulate supercoiling. They play an important role in replication and transcription. Pro T1 Pro T2 Gyrase Eu T1 Eu T2 Cuts 1 strand Cuts 2 strands A bacterial T2 Cuts 1 strand Cuts 2 strands Relaxes –ve Relaxes +ve Introduces –ve Relaxes both Relaxes both supercoils supercoils supercoils +ve and –ve +ve and –ve L# = +1 L#= -2 L#= -2 Passive (uses Uses energy (Only No ATP required Required ATP energy stored in from ATP Introducer) –ve coils) N.B: Reverse gyrase (DNA T1) found in bacteria living at high temperatures can introduce +ve supercoils. This helps stabilize the DNA as the supercoils require energy to relax. Thus, it protects the DNA strand from breakage promoted by high temperatures. The energy released in the breaking of DNA strands is stored as a phosphor-tyrosine bond and is then used to reanneal the strands. 7. What are topological isomers of DNA? DNA differing only in their states of super-coiling. 8. Explain the importance of DNA supercoiling for the cell survival? The only way to get the 2.5m long genome in a nucleus if the DNA is highly coiled. Coiling also prevents access of enzymes to DNA, which prevents unwanted processes from taking place. For example, if DNA were to be unwind at all times, DNAP would make multiple copies of the DNA at all times, costing the cell nutrients and energy and therefore, decreasing its viability. 9. What are the differences between primary (or secondary, or tertiary) structures of RNA and DNA? DNA Primary Secondary Tertiary sugar-phosphate double helical structure structuredouble stranded DNA “chain”with purineand (hydrogen bonding between (both circular AND linear) makes pyrimidinebases as side A-T and G-C; stacking complexes with proteins -supercoil chain(s) interactions; phosphate backbone “outside”) RNA Similar to DNA except RNA molecules frequently Formed through interactions of •2’OH group prevents fold back on themselves to secondary structures: lack of formation of B-helix in 2s form base-paired constraint by long-range regular areas (A-helix is formed) segments between short helices means RNA has high •RNA, like DNA, can be stretches of complementary degree of rotational freedom in single or double stranded, sequences backbone of its non-base paired linear or circular. •G:U= additional, non- regions → capable of folding into •Unlike DNA, RNA can Watson & Crick base pairing complex tertiary structures. exhibit different possible in RNA →enhances *Also, formation of unconventional conformations potential for self U:A:U base triple is possible •Different conformations complementarity in RNA *Pseudo-knots can be formed due (secondary and tertiary •Secondary structures: to base-pairing between sequences structure formation) permit areas of regular helices and that aren’t adjacent. the RNAs to carry o
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