COMPREHENSIVE EXAMINATION

IN

BIOCHEMISTRY AND MOLECULAR BIOLOGY

PAPER #1

                                                                                                Thursday, January 30, 2003

                                                                                                9:00 a.m. to 11:00 am

PLEASE USE THE FOLLOWING INSTRUCTIONS:

1.                  WRITE YOUR NAME OR I.D. NUMBER ON THE UPPER RIGHT HAND CORNER OF EACH PAGE.

2.                  NUMBER ALL PAGES.

3.                  EACH QUESTION SHOULD BE ANSWERED ON A SEPARATE SHEET OF PAPER.

4.                  WRITE THE PAPER NUMBER AND QUESTION NUMBER ON EACH PAGE.

5.                  USE PEN.

6.                  STAPLE PAGES FOR EACH QUESTION SEPARATELY.

PAPER #1

TWO-HOUR QUESTIONS:          

ANSWER ONE OF THE FOLLOWING FOUR QUESTIONS:

Q1.     Regulation of gene expression is known to occur at many different levels. Describe in detail each of these levels and give examples of regulation at that level that occur in prokaryotes and eukaryotes.

OR

Q2.     Using a diagram to simplify your discussion, describe the basic components and mechanisms for transduction of the pleiotypic mitogenic signals via activation of the EGF receptor.

Include in both your diagram and your discussion

a)      A brief description of ligands for the receptor

b)      The basic mechanism for activation of the homodimeric receptor

c)      The basic mechanism for the initial stage of signal transduction through the activated receptor (i.e., the pleiotypic activation of the pathways involved in mitogenesis)

d)     A description of the essential protein components of each step of the central or canonical mitogenic pathway, including class of protein and basic function(s) of each, how each is activated, and the function of each in the transduction of signal which results in altered transcription.

Your diagram should show the ordered recruitment of the pathway proteins into the signaling complex, and your discussion should include a description of how each protein in the pathway is activated.

Q3.    Haniford and Kleckner isolated mutants that affect transposition of a “cut-n-paste” transposon, Tn10, from donor DNA. The way they isolated these mutants is a clever example of how you can take advantage of several known regulatory systems to genetically dissect complex molecular questions. Their approach is shown in the diagram below.

A plasmid carried the Tn10 transposase gene expressed from the P_Lac promoter, which was regulated by the LacI repressor protein. In the absence of IPTG, expression of transposase was turned off, but when IPTG was added high levels of transposase were made. The Tn10 insertion was present on a different replicon that also carried the lacZ gene. The lacZ gene was expressed from the lambda P_L promoter, which was regulated by the lambda cI repressor protein. The cI protein was expressed from a defective lambda lysogen. To answer this question, draw on your understanding of regulation of the lac operon, transposition mechanisms, the lytic/lysogenic switch of phage lambda, DNA break repair, the SOS response, and operon fusions.

a.       Explain the regulation of lacZ expression in cells that express the wild-type transposase. Identify each step in the path and identify the key components acting in each step.

b.      Using the approach of Haniford and Kleckner, how would you isolate transposase mutants that cannot catalyze transposition of Tn10? Explain how would you create the mutations and identify components in the growth medium you would use in your screen/selection.  How would you distinguish the potential mutants from the parent cells?

c.       How could you use this basic approach to isolate dominant-negative transposase mutants that inhibit the transposition activity of wild-type transposase?

OR

Q4.    Industry exerts great effort to develop new antibiotics. One step is to assess human toxicity. For example, when human liver cells were treated with a potential antibiotic called “Compound X” and examined for changes in protein expression, one protein spot on a 2D gel greatly increased in amount. In order to determine if this cellular response to Compound X is a problem, the identity and activity of the induced protein must be determined.

a.    From the spot on the 2D gel, you are provided just enough of a homogeneous preparation of this protein to analyze. How would you deduce the complete amino acid sequence of this protein?

b.   Given the amino acid sequence, how would you determine the function of this protein? Describe the steps you would take in sufficient detail to satisfy your group leader that you are on the right track.

c.   In your analysis, you determined that the protein is a member of a previously uncharacterized protein family with at least one domain common to a class of dehydrogenases that use an NAD/NADH cofactor. Many of the related dehydrogenases act on one of a few amino acid substrates. Describe the steps you would take IN DETAIL to determine if the unknown “spot protein” is an NAD-dependent amino acid dehydrogenase. Include the type of assays you would employ, the equipment required to run the assays, and the form the data would take.

d.   Given that you have confirmed that NADH is a cofactor and is converted to NAD during the reaction, describe how you will determine Km and Kcat of the enzyme for the amino acid substrate(s). How would you use this kinetic information to determine the most plausible amino acid substrates?

                                                                                                (END OF PAPER #1)
 

COMPREHENSIVE EXAMINATION

IN

BIOCHEMISTRY AND MOLECULAR BIOLOGY

PAPER #2

                                                                                                Thursday, January 30, 2003

                                                                                                1:00 p.m. to 3:00 p.m.

PLEASE USE THE FOLLOWING INSTRUCTIONS:

1.      WRITE  YOUR NAME OR I.D. NUMBER ON THE UPPER RIGHT HAND CORNER OF EACH PAGE.

2.      NUMBER ALL PAGES.

3.      EACH QUESTION SHOULD BE ANSWERED ON A SEPARATE SHEET OF PAPER.

4.      WRITE THE PAPER NUMBER AND QUESTION NUMBER ON EACH PAGE.

5.      USE PEN.

6.      STAPLE PAGES FOR EACH QUESTION SEPARATELY.

ANSWER ONE OF THE FOLLOWING TWO QUESTIONS (60 MINUTES)

Q5.     Describe in detail the steps and enzymes involved in the excision of uracil from duplex DNA and the subsequent steps of base excision repair in E. coli. Choose an enzymatic step in this process and describe a quantitative assay (including the overall principle that you are testing and the substrates and other cofactors that would be necessary as well as all appropriate controls) that you would use to measure this activity. What would be the consequence following two rounds of replication if uracil were not removed? Where appropriate, use diagrams to illustrate your point.

OR

Q6.      Discuss the mechanisms of ATP-dependent chromatin remodeling. Include the following: the structural changes in nucleosomes (or nucleosomal arrays) during remodeling, the type of motif in the catalytic subunit of ATP-dependent chromatin remodelers, and current views on how remodelers catalyze structural changes. Also describe one of the many assays used to monitor chromatin remodeling and the type of structural change it detects.

ANSWER ONE OF THE FOLLOWING TWO QUESTIONS (30 MINUTES)

Q7.     Describe how genetic analysis in yeast provided evidence for the existence of “checkpoint controls”, and give at least two examples of checkpoint controls that operate during the eukaryotic cell cycle.  What do you expect to be the underlying mechanism for how checkpoints function?  How might drugs that interfere with checkpoint controls be useful in cancer therapy?

OR

Q8.      Compare the mechanism of DNA replication initiation in prokaryotes and eukaryotes highlighting the differences and similarities where they exist.  Include in your discussion the following topics, (1) origin structure and function, (2) initiator proteins, (3) the role of DNA polymerases I and III (in bacteria), and Pol a, d, and e (in eukaryotes). 

ANSWER ONE OF THE FOLLOWING TWO QUESTIONS (30 MINUTES)

Q9.    Provide three examples of regulatory regions in the 3'UTR of an mRNA and what their functions are.

OR

Q10.  Alternative pre-mRNA (mRNA precursor) splicing is caused by flexibility of 5' or 3' splice sites selection, and multiple mRNA molecules are generated from one gene transcript.  Describe its biological significance on the regulation of gene expression.

ANSWER ONE OF THE FOLLOWING TWO QUESTIONS (30 MINUTES)

Q11.  The previously identified gene for eRF1 was placed on a plasmid and expressed in three eRF1-minus strains, a, b, and c. of the fungus I. imaginarius.  This simple eukaryote does not undergo splicing.  It uses the standard genetic code (copy attached) and has the standard 20 amino acids.  The protein found after expression was unexpected based on the DNA sequence.  Relevant parts of these sequences are shown below.  The upper line is the gene sequence, the next three lines are the amino acid sequences for the protein found in a, b, and c.  (1) What is the nature of the unexpected results?  (2) How do you account for them?  (3) If there is more than one possible explanation for each result, indicate how you would distinguish among them.

....GUU CUU AGG GGG TAT CTT TAG CTA CGA CGC CAA GAA AGA....

a....V   L   R   G   Y   L   Q   L   R   R   Q   E   R.....

b....V   L   R   G   Y   L   L   A   T   T   P   R   K.....

c....V   L   R   G   Y   L   S   Y   D   A   K   K   E.....

OR

Q12. 

1. Mature mRNAs for almost all proteins contain a number of AUG sequences.  (1) How does the translational machinery in the cell know which AUG to use for initiation of protein synthesis?  (2) How does it choose AUG instead of some other codon?  Compare and contrast the process in prokaryotes and eukaryotes in as much detail as possible. 

2. In order for an amino acid to be inserted in a growing peptide chain, two fundamental reactions have to occur at each addition of an amino acid.   One occurs on the small subunit and one on the large subunit.  What are they?  Describe each process in detail.

3. Why is puromycin an effective inhibitor of protein synthesis?

4. What two critically important roles essential for protein synthesis does attachment of an amino acid to its tRNA do?

(END OF PAPER #2)

COMPREHENSIVE EXAMINATION

IN

BIOCHEMISTRY AND MOLECULAR BIOLOGY

PAPER #3

                                                                                                Friday, January 31, 2003

                                                                                                9:00 a.m. to 11:00 a.m.

PLEASE USE THE FOLLOWING INSTRUCTIONS:

1.      WRITE  YOUR NAME OR I.D. NUMBER ON THE UPPER RIGHT HAND CORNER OF EACH PAGE.

2.      NUMBER ALL PAGES

3.      EACH QUESTION SHOULD BE ANSWERED ON A SEPARATE SHEET OF PAPER

4.      WRITE THE PAPER NUMBER AND QUESTION NUMBER ON EACH PAGE.

5.      USE PEN.

6.      STAPLE PAGES FOR EACH QUESTION SEPARATELY.

ANSWER ONE OF THE FOLLOWING TWO QUESTIONS (30 MINUTES)

Q13.   Describe the fundamental principles that give rise to the initially detected electrical signal (FID) in an NMR experiment.

OR

Q14.  A wide range of approaches has been developed to characterize nonnative states of proteins in atomic detail by NMR spectroscopy, and evidence for the persistence of native-like structure even under strongly denaturing conditions has been reported.

a)      List and describe two different NMR-measurable parameters that indicate that the backbone amide group of a given residue or stretch of residues in a protein behaves either as a random coil or is restricted to a given residual structure.

b)      Describe how enthalpy and entropy could contribute to the observed persistence of long-range structure under denaturing conditions.

ANSWER ONE OF THE FOLLOWING TWO QUESTIONS (30 MINUTES)

Q15.  You have isolated a protein and know its sequence. How would you quickly determine its helical content. Explain the techniques you use and discuss their caveats.

OR

Q16.  Discuss the structural and physical basis of cooperativity in the folding of proteins and nucleic acids.

ANSWER ONE OF THE FOLLOWING TWO QUESTIONS (30 MINUTES)

Q17.          

A.    You have decided to use a nitrocellulose filter-binding assay to monitor fractions during purification of a DNA-binding protein. Describe a method to produce a ³²P­labeled duplex DNA probe. Quantitation of the probe by scintillation counting shows that

B.     1 µl (10 fmol) contains 1500 cpm. Assuming that scintillation counting is 100% efficient (i.e. cpm = dpm and 1 Ci = 2.2 x 10¹² dpm), what is the concentration (mol/1) and specific activity (Ci/mol) of your probe?

C.     Using the nitrocellulose filter-binding assay you have purified the protein to near homogeneity. Titration of the purified protein in the nitrocellulose filter-binding assay shows that 0.1 tl of your protein solution leads to retention of 10,000 cpm of your ³²P­labeled DNA probe. Given the specific activity of your probe, how many mol were retained?

D.    The following table shows the absorbance at 595 nm of given volumes of a protein standard at 0.2 mg/ml in the Bradford dye-binding method for protein determination

Volume          Absorbance

   10                       0.05

   25                       0.1

   50                 0.2

 100                  0.4

5 ml of your purified protein gives an absorbance of 0.3. What is the concentration (mg/ml) of your sample?

OR

Q18.  The classical Michaelis-Menten equation for enzyme kinetics is found to have a broader applicability than just the case of enzyme catalysis. For example, the rate of transport of a molecule across te cell membrane by a specific transport molecule can be described by the same equation. Illustrate with a diagram/model how this can be. In the case of the Michaelis-Menten equation, certain assumptions had to be made in order to derive the equation. What form would these assumptions now take in the case of active transport?

ANSWER ONE OF THE FOLLOWING TWO QUESTIONS (30 MINUTES)

Q19.  If a macromolecule is placed in a centrifugal field, what factors will determine the rate at which the molecule moves through the solvent? If we observe a protein solution stored in the refrigerator for one year during which time there has been a continuous force of 1G, we find that the protein is uniformly distributed throughout the container – how can this be?

OR

Q20.   It is commonly observed that the molecular mass of a protein determined by gel filtration is very different than that determined by glycerol gradient centrifugation. Provide an explanation for this phenomenon and suggest a method for mass determination that would not be subject to these discrepancies.

(END OF EXAM)