Exam 3, BICH 410 (TuTh 12:45-2), Tuesday, Nov. 19, 1996

Write your name on each page. Write concise answers to demonstrate effectively your mastery of the subject material. Show your work in order to receive partial credit where applicable.

1. (6 pts) Circle the double stranded DNA in each pair that you would expect to have the highest melting temperature.

A) 1000 bp of 30% G+C composition in 10 mM NaCl OR 1000 bp of 50% G+C composition in 0.1 M NaCl
Answer: 1000 bp of 50% G+C composition in 0.1 M NaCl; higher %GC and higher salt

B) 500 bp of 70% A+T composition in 0.05 M NaCl OR 500 bp of 20% G+C composition in 0.05 M NaCl
Answer: 500 bp of 70 A+T composition in 0.05 M NaCl; higher %GC (30% vs. 20%)

C) 20 bp of 50% G+C composition in 10 mM NaCl, 1M urea OR 20 bp of 50% G+C composition in 10mM NaCl, 10M urea
Answer: 20 bp of 50% G+C composition in 10 mM NaCl, 1M urea; lower [urea] means higher melting pt.

2. (9 pts) In a supercoiled DNA, a segment of 252 base-pairs changes from the B-form to the Z-form without ever breaking the phosphodiester backbone. Assume that the DNA remains entirely double-stranded. What is the change in (remember to include the sign (+ or -)):

(A) the twist, T?
Answer: lose 252 bp / 10.5 bp/turn = 24 positive turns; gain 252 bp / 12 bp/turn = 21 negative (left-handed) turns; therefore T = -24 + (-21) = -45

Answer: Lk = 0 because the phosphodiester backbone was never broken.

(C) the writhe, W (supercoiling)?
Answer: Lk = T + W; since Lk = 0, then W = -T; so W = +45

3. (4 pts) Given the following sequence for an RNA molecule, draw a secondary structure that will be maximally stable. Clearly indicate the bases that are paired. (Hint: Ignore G-U base-pairs.)
5'-GUCCAGCCAUUGCGUUUCGCAAUGGC-3'

Answer: see posted key; the RNA forms a stem-loop structure with intramolecular base-pairing; nine base-pairs can be formed separated by 3 nts. in a loop

4. (10 pts) You are trying to isolate a cDNA clone that encodes a certain protein. The amino acid sequence of a peptide obtained from this protein after cleavage with trypsin is:
thr-ala-tyr-val-leu-gly-trp-cys-gln-met-trp-phe-pro-ala-val-ser-arg
Using the genetic code reproduced on the last page of this examination, design an oligonucleotide DNA probe of length 20 nucleotides that could be used to screen your cDNA library. Your answer will not be a unique sequence of 20 nucleotides. How many different 20-mers will have to be synthesized to ensure a perfect complementary match to the real gene sequence?

Answer: Use the genetic code to determine the region of least degeneracy. Figure the number of codons possible for each amino acid. The region of the peptide from trp-cys-gln-met-trp-phe is encoded by 1-2-2-1-1-2 codons, respectively, which would be 18 nt. long. The next codon, pro, always starts with CC (no variability in first two positions), so the nucleic acid sequence could be increased to 20 nt. without adding any more degeneracy. The sequence of a 20-mer (with variations) would be:
5'-TGGTGT(C)CAA(G)ATGTGGTTT(C)CC-3'
At three positions there are two possible nucleotides, so 2 x 2 x 2 = 8 different oligos would need to be synthesized to cover all possibilities.

5. (10 pts) Given the following double-stranded fragment of DNA:
5'-CCTGCAGGTTAGCTGCTTACGGGCAATCGTATTGCCGTAC-3'
3'-GGACGTCCAATCGACGAATGCCCGTTAGCATAACGGCATG-5'
Using the oligonucleotide primer, 5'-CGATTGCCCGTAAGCAGC-3', write the sequence of ALL the DNA fragments that would be generated by the Sanger (dideoxy) sequencing method in the samples that contain (A) ddATP, and (B) ddGTP. Clearly show your reasoning in order to receive partial credit.

Answer: The primer anneals perfectly to the top strand in an antiparallel orientation:
DNA polymerase synthesizes new DNA from the 3' end of the the primer in the 5' to 3' direction. After incorporating a dideoxy nucleotide in the newly-synthesized DNA (same strand as primer), synthesis of that molecule is terminated.
(A) In the presence of ddATP, the following fragments are produced:
5'-primer-TA-3'
5'-primer-TAA-3'
5'-primer-TAACCTGCA-3'
5'-primer-TAACCTGCAGG-3' (run-off)
(B) In the presence of ddGTP, the following fragments are produced:
5'-primer-TAACCTG-3'
5'-primer-TAACCTGCAG-3'
5'-primer-TAACCTGCAGG-3'

6. (31 pts) Answer the following questions. (A) A wax consists of a long-chain __________________ and a long-chain _____________ in a(n) ___________ linkage.
(B) Which is more soluble in water? (Circle the correct answer) stearic acid (an 18:0 fatty acid) OR a simple triacylglycerol containing stearic acid
Answer: stearic acid is more soluble because contains a charged carboxylate group, whereas the carboxyls are all esterified in the triacylglycerol
(C) What enzyme cleaves inositol triphosphate from the membrane phospholipid?
(D) What enzyme is used to copy messenger RNA into DNA? ____________________. This enzyme is necessary to construct a ____________ library.
(E) A bacterial enzyme that can introduce negative supercoils into DNA _________________
(F) Extended length of DNA (in Å or nm) in a haploid yeast genome (1.3 X 107 bp)
Answer: (1.3 X 107 bp)(3.4 Å/bp) = 4.4 X 107 Å; or 4.4 X 106 nm
(G) What is the name or structure of a ketose containing three carbons? _________________
(H) What type of end predominates in a molecule of glycogen (circle the correct answer)?
reducing end OR non-reducing end OR same number of each type
Answer: non-reducing end; branched structure means many non-reducing ends and one reducing end
(I) What type of end predominates in a molecule of amylose (circle the correct answer)?
reducing end OR non-reducing end OR same number of each type
Answer: same number of each type; amylose is not branched
(J) What is the chemical difference between a nucleoside and a nucleotide?
Answer: nucleotide contains a phosphate(s) esterified to the carbohydrate whereas the nucleoside contains just the base and the carbohydrate
(K) In one or only several words, each, list the steps involved in one cycle of PCR in the order that they must occur.
1) denaturation of DNA
2) annealing (hybridization of primers (oligonucleotides)
3) extension; synthesis of DNA from primer by DNA polymerase in presence of dNTPs
(L) Name of histone that binds the linker DNA between nucleosomal core particles _________