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University of Waterloo
BIOL 308
Dragana Miskovic

Study questions for the lectures 5-7 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. 2. How does complexity of bacterial genome differ from that of eukaryotic (calf) genome? • Cot ½ of calf DNA is greater than the Cot ½ of E. coli DNA (due to larger genome size) • Bacterial (E. coli) genome has no repetitive DNA sequences (genome is one unique sequence), whereas eukaryotic (calf) genome consist of 3 classes of DNA sequences: unique (single copy), moderately repetitive, and highly repetitive DNA sequences • In E. coli, it is initially difficult for sequences to find their complements during renaturation; however, once they are found, there is fast re-association (zippering) • In calves, there is fast renaturation of the highly repetitive sequences, then middle renaturation of the moderately repetitive sequences, and then slow renaturation of the unique sequences • The difference in renaturation patterns of E. coli and calf genome give them 2 different Cot curves (E. coli DNA resembles an ideal Cot curve while the calf DNA curve is not smooth) 3. Explain C value paradox. • There is no correlation between the amount of DNA (size of genome) and the apparent complexity of organisms • Ex. some single celled protists (ex. Amoeba) have genomes much larger than that of humans, but this doesn’t mean that Amoeba are more complex than humans 4. List and briefly explain factors that influence DNA renaturation kinetics. • DNA concentration: complementary single strands must find each other; when DNA concentration is low, the single strands will have a hard time finding each other and renaturation will be slow; however, too much DNA may not be ideal either (there is an optimal DNA concentration at which renaturation happens the fastest) • Salt concentration: ionic conditions (+ve ions) mask the repulsive forces of (-ve) phosphate backbones of the single stranded DNA and thus speeds up renaturation (calculate optimal concentration of salt to add to solution to prevent repulsion b/w the single stranded DNA) • Temperature: must slowly lower the temperature (to 20-25°C below melting temperature (T m) to allow the single strands to find each other and bind to their complements • Time (reaction time): given enough time (which varies depending on the DNA molecules), the single strands will eventually find their complementary sequences and bind • Size of the DNA fragment (inversely proportional): the smaller the fragment, the greater the probability they will find their complementary sequence; the larger the fragment, the lower the probability they will find their complementary sequence • Complexity: simple (repetitive) sequences renature faster than complex (unique) sequences 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? • 3 classes of DNA: highly repetitive, moderately repetitive, and unique sequences (b) What can you say about the relative complexity of each class? What fraction of the genome falls into each class? • The first 25% of DNA that is renatured are the highly repetitive sequences • The middle 25% of DNA that is renatured are the moderately repetitive sequences • The last 50% of DNA that is renatured are the unique sequences 6. List three (3) differences between prokaryotic Topoisomerase I and Gyrase. (L: 6, S: 24) Topoisomerase I Gyrase (bacterial Topo II) Relaxes negative supercoiling Relaxes positive supercoiling Does not use ATP Uses ATP Changes linking # in steps of 1 Changes linking # in steps of 2 7. What are topological isomers of DNA? (L: 6, S: 16) • DNA differing only in their states of supercoiling (DNA sequence is the same) 8. Explain the importance of DNA supercoiling for the cell survival? (L: 6, S: 16-17) • Important for “packing” of DNA in the nucleus; one way of making DNA more condensed • Supercoiling also plays a role in replication and transcription by reducing the induced stress by twisting the double helix – this is done to provide a single- stranded template for synthesis of a new complementary strand (it is also energetically cheaper to bend long DNA than to untwist it) 9. What are the differences between primary (or secondary, or tertiary) structures of RNA and DNA? • Primary o RNA has a 2’ OH group which prevents the formation of B-helix (A-helix is formed) o DNA has a 2’ hydrogen which allows for the formation of B-helix o RNA contains uracil in place of thymine (which is present in DNA) • Secondary o DNA has a double helical structure (H-bonding between A-T and G-C; stacking interactions; phosphate backbone facing the “outside”) o RNA molecules frequently fold back on themselves to form base-paired segments (and can form stem-loop and hairpin structures) • Tertiary o DNA: can make complexes with proteins to undergo supercoiling o RNA: has high degree of rotational freedom in backbone of its non-base- paired regions (formation of unconventional U:A:U or A:G:C base triple is
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