Undergraduate students should select any seven problems and provide careful, detailed, well-worked out answers. Graduate students select eight problems.

• Some problems are short and relatively easy, others require more extensive work. Provide thorough and complete answers; no question should require more than one page for a satisfactory answer, but I will not complain about additional length if the content is of high quality.

• Be sure to cite any resources other than class notes that you use in preparing your answers. Do NOT simply copy something from a book or paper; use your own words.

• When I suggest a paper as a starting point, do NOT use it as your only resource!

• Some of these problems will seem kind of open-ended; they are intended to be. What I want you to do is pick something you think you'd like to know more about, and check it out.

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 sequence of a segment of E. coli DNA (from the middle of the coding region, so no start codon is present) is:

5'-CCGGCTAAGCCATGACTAGC-3'
3'-GGCCGATTCGGTACTGATCG-5'

Write the sequences of the two mRNAs that could be transcribed from this region. Then write the amino acid sequences that would be coded by these two mRNAs. Finally, choose an alternative reading frame, and repeat the process. (BioEdit can be used in this problem, if you wish.)

3. 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.

4. 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?

5. 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.

6. 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.)

7. 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. 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.

8. 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.

9. 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. A recent paper describes a possible abiotic synthesis of ribose, as a starting point for the appearance of ribonucleotides on primitive earth. Provide a clear, careful description of how this could occur. A reasonable starting point would be: Science, 2004, 303, 196 A. Ricardo, M. A. Carrigan, A. N. Olcott, S. A. Benner).

10. 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 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.

11. 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.

12. A "dotplot" is a graphical representation of a sequence alignment. It is a matrix in which the rows correspond to the bases (or amino acids) of one sequence, and the columns to the bases of the other sequence. In its simplest form, a dot is placed in a matrix element if the two bases defining it are the same. For exactly identical sequences this would produce a diagonal line, with scattered points elsewhere.

Retrieve from GenBank the gene sequence of Mitochondrial ATPase subunit 6 from the Atlantic hagfish (Myxine glutinosa), and for the same gene from sea lamprey (Petromyzon marinus) or subunit 6 of any other ATPase. Investigate their similarity using BioEdit to create both a direct alignment and a dotplot. (NOTE: If you downloaded a version of BioEdit later than v. 9.0.4, the dot plot option is no longer available. Use this web site instead, pasting your two sequences into the two boxes offered.)