Study questions for required reading.

Study questions for Watson and Crick, 1953:

1. Does the WC structure most clearly resemble A, B, or Z form? Why?
2. The authors state that "the two chains (but not the bases) are related by a dyad perpendicular to the fibre axis." What is a "dyad," and what does this statement suggest about whether the chains are parallel or antiparallel? Under what circumstances would the bases in fact be related by a true dyad axis?
3. Draw the preferred enol form of thymine (why is it preferred?). What would enol-T base pair with? Why was it important to Watson and Crick that the keto forms of the bases be preferred?
4. Identify a nomenclature/numbering inconsistency between the WC paper and today's labeling.
5. The authors specifically suggest that their helical structure cannot apply to RNA. Where/how do they say this?

Study questions for hybridization thermodynamics:

1. Download the oligonucleotide hybridization spreadsheet. Learn to use Microsoft Excel if necessary.You may also want to look at the underlying math, or not..
2. In the spreadsheet, change ΔS° and ΔH° and observe effects on the melting curve. Change both ΔH° and ΔS° at the same time but try to keep the overall stability, roughly expressed by
   ΔG°₃₇ = ΔH° -310ΔS°
   constant. What happens to the sharpness of the transition as ΔH° and ΔS° increase?
3. Qualitatively guess at how accurately ΔH°, ΔS°, ΔG°₃₇ and T_M can each be measured from experimental data. Give your reasoning based on the results from above.

Study questions for Moser and Dervan, 1987:

1. Why do we focus on these particular triple base pairs instead of any of the legion of other possibilities? (Remember what’s important about the particular Watson-Crick base pairs vs. all the others.)
2. Why are the triplexes studied here stabilized by low pH? Why are they stabilized by polyamines? Why is the increased stability of triplex at acidic pH not apparent in Figure 5?
3. Figure 3 demonstrates parallel orientation of the third strand oligo-T and the homopurine strand in the duplex. What is the reasoning leading to this conclusion? How does it rule out strand displacement as a mode of binding?
4. If the third strand probe bound in the minor groove rather than the major groove, what would the DNA-EDTA 9 footprinting histogram on the bottom right of figure 4 look like, and why?
5. Why do the shorter probes or mismatches in Fig. 4 cleave reasonably well at low temperature but then cleave (and presumably bind) less and less effectively as the temperature is increased?
6. Why has it not been possible to generalize triple-strand recognition to double-stranded targets of arbitrary sequence? In other words, why the restriction to homopurine/homopyrimidine tracts?

Study questions for the chimp genome draft sequence, 2005.

This is a very long and complex paper, though very well-written. You do not need to understand every detail, but you are responsible for the starred (*) study questions. You need not read the Methods, but the short Discussion section is important. You may need to use Google or Wikipedia to answer some of these questions if too many terms are unfamiliar. Warning: Since this is not my field, some of the answers provided may be simplistic.

1. *What is “sequence redundancy?” Why is it necessary to have significant redundancy in order to assemble a genome using whole-genome shotgun sequencing (WBS)?
2. What's the difference between nucleotide-level accuracy and structural accuracy? What is the idea behind the claim that the substitution rate assessed by comparison with a BAC is about what one would expect because the BAC is a single haplotype?
3. Figure 1b shows the divergence between chimp and human. How was the figure constructed, and what do the individual symbols mean?
4. The authors show that sequence divergence is much more rapid at CpG sites, due to cytosine methylation (at position 5) and then deamination. What would be the product of methylation/deamination, and why does that lead to more mutation than other kinds of DNA damage?
5. Is there a simple bottom line for the cause of the variation in divergence frequency across chromosomes?
6. *What are “indels” and how do they ay arise?
7. Figure 7 shows that old Alu mobile elements are more likely to be found in GC-rich regions. What explanation do the authors offer?
8. *What are “purifying selection” and “positive selection,” and how are they reflected in the Ka/Ks ration?
9. Table 3 shows that rare alleles at human polymorphic loci are more likely to have changes in coding sequences than common alleles or between human and chimp. The authors suggest that this reflects the genetic load of mutation. What does this mean?
10. *What’s the point of looking closely at genes that have diverged more rapidly than other genes?
11. What does the dramatic dip in the middle of Figure 10 mean?
12. Figure 12 suggests that transcription factors are one class of genes that have diverged more rapidly in the evolution of humans than of chimpanzees. If true, what does this suggest about sources of phenotypic changes? Why isn’t the TF point an obvious outlier on Fig. 12?
13. *On page 81, the authors try to identify which human SNPs are ancestral and which are new by comparison with the chimp as an “outgroup.” What’s the principle behind this idea?
14. What does the slope of Figure 13 mean?
15. *How does reduced diversity relative to divergence suggest a selective sweep in human history?
16. *The discussion, and in fact most of the paper, focus on distinguishing adaptive change from neutral drift or even changes to less-fit phenotypes. Which type of change is responsible for the bulk of the observed changes? Why have maladaptive changes apparently been maintained more frequently in hominids than rodents? Is there a possible upside to this genetic load?

Study questions for simple protein-nucleic acid binding curves

Create a spreadsheet to study the behavior of the simple equation Y = P/(P+Kd).
1. Create a column for protein concentration and one for fraction bound, assuming negligible nucleic acid concentration. Graph the results. Try a linear vs. a log scale on the x axis. If you were doing an experiment, how would you space the choice of protein concentration? (For example, if you think Kd is about 10 nM, would you test [P] = 0,2,4,6,8,10,12,14,16 nM or might you be better off testing 0,0.5,1,2,4,8,16,32,64 nM?) What is the range of concentration over which binding goes from 10% to 90%? Does the ratio between these limits depend on Kd? By analogy with myoglobin/hemoglobin, what might it mean if you observed a much sharper dependence on concentration?
2. How would you measure Y for an electrophoretic mobility shift assay? How about a quantitative DNAseI footprint? Consider issues of background correction and incomplete protection.

Study questions for Seeman et al., 1976:

1. Why is it likely to be difficult for proteins to use all six of the major groove recognition sites W1, W2, W3, W1’, W2’, and W3’ for sequence-specific recognition?
2. Why is the Table I entry for (G-C/C-G) discrimination at the S2’ position a “(0)”? In other words, what is the basis for discrimination between the two different base pairs, and why is it likely to be difficult?
3. Inosine, which is the same as guanine except that the 2-NH2 group is replaced by H, can be used as a probe for the groove recognized by a protein. Compare the I-C base pair to the A-U and G-C base pairs and predict the result of an experiment where G is substituted by I and the binding of either a major-groove binding protein or a minor-groove binding protein is studied.

Study questions for Ren et al., 2000:

1. What is LM-PCR, the method by which IP-enriched DNA was amplified? Before you look it up, think about what it must do. Why couldn't they amplify the IP'd DNA with regular old PCR?
2. What is the point of Figure 1B? What do the red and blue dashed lines mean? Why do they flare out at the bottom left? What's a P value?
3. Does it strike you as odd that the differences in expression levels in Figure 2 are so much larger than the differences in occupancy levels? Similarly, GAL4 is observed to bind at only a few of its many recognition sites in the genome. What is the likely explanation for both observations?
4. The MTH1, PCL10, and FUR4 genes could have been identified as being up-regulated by GAL4 simply on the basis of mRNA expression data. Why does the ChIP-chip method still tell us something we wouldn't have known only from expression data?
5. Why are all the rightmost lanes in Figure 2B blank?
6. What other types of DNA binding proteins (besides transcription factors) would be interesting to examine using this method?

Study questions for Naktinis et al., 1996

1. What is the basis of the “protein footprinting” assay used to measure interactions among core, beta, and gamma?
2. What is the evidence for competitive as opposed to simultaneous binding of core and gamma to the beta sliding clamp?
3. Figure 7 shows that the gamma complex can remove sliding clamps from DNA, but the evidence is that the +gamma curve changes very little with time. Why is much of the clamp still bound at the end of the reaction? What might the gel filtration profile have looked like in the presence of a large excess of linear DNA?
4. What is the advantage to the cell in having primer-template DNA but not nicked DNA stabilize the beta-core interaction? How does this play out during the synthesis of an Okazaki fragment?

Study questions for Cosma et al., 1999

1. In Figure 1, what is the evidence for specific binding of Swi4p to the URS2 region? What do the different "delta" lanes in 1B mean? Why do the authors do a dilution series of the WCE (= whole cell extract) as a control?
2. You do not need to understand all the tricks used to manipulate and monitor the yeast cell cycle, but one of the main points of the paper is the use of cell-cycle regulated genes as a good model system for following the order of events at a promoter. Why is this important? Why couldn't the authors just do their experiments with an unsynchronized population of cells? How else might one follow the order of events at a regulated promoter? In figures 2 and 3, FACS (= fluorescence-activated cell sorting) is a way to measure the DNA content of individual cells in a population. It is used to help show that Ash1p prevents binding of Swi4p to the URS2 during the first cell cycle after release from a block and that HO mRNA and Swi4p binding are cell-cycle dependent.
3. What is the evidence that at this promoter the action of SWI/SNF is necessary to recruit SAGA (which includes HAT activity)?
4. The authors note that Swi5p is present on DNA only transiently but leads to long-lasting effects. They showed this using ChIP assays on carefully synchronized cells. On page 306 they discuss the evidence that this observation is real and not due to "epitope masking." What would epitope masking do to the ChIP assay? If it were actually occurring, what would be the resulting hypothesis as to the role of Swi5p?

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