Undergraduate students should select any seven problems and provide careful, detailed, well-worked out answers. Graduate students select eight problems. I prefer problem sets to be word processed and sent to me electronically. If you must use dead trees, BE NEAT!
1. (a) As we described in class with a cartoon, single stranded RNA often forms hairpin turns that allow it to base pair with itself. Here is the sequence of one such strand, reading from the 5' end: CUAGAUGGUAGGUACGGUUAUGGGAUAACUCUG. Suggest how this strand might form a hairpin. That is, which bases would pair, and which would be in the turn? More than one answer is possible. Again, a graphic would be the best way to answer this.
(b) The Mfold server will make a prediction of the folding of any segment of RNA or single-stranded DNA. Submit the sequence above to this server. Compare your prediction to that of the server and comment on any differences.
2. The nuclear genome of the wooly mammoth has recently been sequenced [Miller et al., Nature, 2008, 456, 387]. Discuss this work, including the source of the DNA, the methodology, and the significance of the results.
3. (a) Some naturally occurring polynucleotide sequences are palindromic; that is, they are self-complementary about an axis of symmetry. Such a sequence is:
---TCAAGTCCATGGACTTGG----
---AGTTCAGGTACCTGAACC----
Such sequences can form a double hairpin, or cruciform, conformation. (Think of the shape if you push toward the center on each end of this sequence, and one strand bulges upward while the other bulges downward.) Show how this sequence might form such a conformation, showing clearly the symmetry and self-pairing.
(b) Restriction endonucleases are enzymes that cleave both strands of DNA duplexes at specific sites. The recognition sites for these enzymes often are palindromic.
Here are two palindromic DNA sequences:
5'-G-A-A-T-T-C-3'
3'-C-T-T-A-A-G-5'
5'-G-A-T-A-T-C-3'
3'-C-T-A-T-A-G-5'
The palindromes can be found by carefully placing a two-fold symmetry axis between the strands, perpendicular to the page.
(a) Locate the palindromes and the symmetry axes.
(b) The EcoRI enzyme cleaves the first sequence between G and A. Identify the cleavage points. The resulting fragments are said to have "sticky ends". What does this mean?
(c) EcoRV cleaves the second sequence between T and A. Identify the cleavage points. The resulting fragments have "blunt ends". What does this mean?
4. Restriction endonucleases were first discovered in bacteria, where they restrict the growth of viruses by cutting foreign DNA. As noted in problem # 4, EcoRI looks for the sequence GAATC. How is it that the bacteria do not cut their own DNA when this sequence is encountered? Some references that may help: (1) Meselson M., Yuan R., "DNA restriction enzyme from E. coli", Nature, 1968, 217, 1110-4; (2) Wilson G.G., Murray N.E., "Restriction and Modification Systems". Annu. Rev. Genet., 1991, 25, 585-627; (3) Kessler C., et. al. "Recognition sequences of restriction endonucleases and methylases", Gene, 1985, 33, 1-102.
5. BioEdit is a very useful tool for many manipulation of DNA, RNA, and protein sequences. One of the simplest capabilities is conversion of a DNA sequence into the protein sequence for which it codes. Search GenBank for the DNA coding for the laccase enzyme from the fungus Trametes versicolor. (A tutorial for GenBank is included on this Page.) Use BioEdit (or other software of your choice) to translate it into an amino acid sequence. Tabulate the frequencies of occurrence of the bases in the DNA and of the amino acids in the protein. (A tutorial for BioEdit is HERE.)
6. The classic Watson/Crick paper we referred to in class calls attention to an earlier paper by Linus Pauling and Robert Corey that presents an incorrect suggestion for the structure of DNA. Read that paper. What structure did Pauling propose? Could it possibly be consistent with the Meselson-Stahl experiment? Or with the A/T and C/G ratios of 1? Be specific and complete in your analysis.
7. Shown below is the crystal structure of E. coli helicase (ribbons) complexed with a segment of DNA (space-filling), as determined by Korolev, et al., Cell, 1997, 90, 635. Using that paper as a starting point, explain in as much detail as possible, how the enzyme unwinds the DNA.
8. Many scientists believe that life on earth started in an "RNA World". That is, RNA preceded both DNA and proteins as the genetic medium and the catalytic medium for life processes. In this view, the large amount of noncoding RNA produced from our DNA is a relic of this early world. Read the paper by Joyce, Nature, 2002, 418, 214 and discuss some of the evidence for RNA world and how the transition to DNA world might have come about.
9. In 1980, Frederick Sanger received his second Nobel Prize in chemistry for a method of sequencing DNA. (His first Prize, also in chemistry, was for a method of protein sequencing.) Explain how the Sanger method worked, and how the modern modification of it using fluorescent tags works. Here is a starting point, Sanger's original publication of the method: Proceedings of the National Academy of Sciences of the USA, 1977, 74, 5463.
10. We mentioned in class a "Y-chromosomal Adam". Summarize the evidence supporting this idea. Here is a starting point: Gibbons, Ann; "Y Chromosome Shows That Adam Was an African." Science, 1997, 278, 804-805.
11.Gene duplication has been recognized as an important part of evolution since the 1930s. Read these papers: Zhang, Trends Ecology Evol., 2003, 18, 292; Hurles, PLoS Biology, 2004, 2, 0900. Discuss how gene duplication occurs and what can happen to a duplicated gene.
12. A phenomenon called "gene silencing" has been discovered recently. It involves small interfering RNAs binding to mRNA and tagging it for destruction before it can be translated into protein. Provide a careful, complete description of how this genetic control works. Starting points: Nature, 2002, 418, 244; Nature, 2004, 430, 161; Nature, 2004, 431, 338; Science, 2002, 296, 1260ff.
13. Do problem #13 at the end of Chapter 28 in Lehninger. Include with your answers pictures to help illustrate them. The web page explains how to capture images.
For those working with the 4th edition: To examine the roles of hydrogen bonds and hydrophobic interactions between transcription factors and DNA, go to FirstGlance: http://firstglance.jmol.org. Enter the PDB id 1tgh in the query box.
This file contains the crystal structure of a human TATA-binding protein and a segment of double stranded DNA.