Distinguish in biochemical detail between ?-oxidation of saturated and unsaturated fatty acids

Look up and list the main properties of the acids formed in acid rain.

Compare and contrast the difference between Proteins, Carbohydrates, Lipids and Nucleic acids?

What precautions should you take when working with strong acids?

You are responsible for characterizing a novel mutant version of PAH from a patient experiencing mild symptoms of the disease. You first task is to purify the enzyme responsible. You decide to generate a construct to express the protein in E.coli.

Step 3-1: PCR to amplify the mutated gene. You have the following samples: a. Genomic DNA from the patients hepatocytes. b. Genomic DNA from the blood cells. c. mRNA from the hepatocytes d. mRNA from the blood cells e. cDNA from the hepatocytes f. cDNA from the blood cells. Which would you choose as your template and why?

Step 3-2: Creating a construct. Which sequence elements would your DNA construct require for expression in E. coli to be successful?

Circle all that apply. Bacterial origin of

replication (ORI) Human ORI

Human TATA box sequence Explain:

Bacterial Ribosome Binding Site (RBS)
Human (RBS)

Antibiotic resistance gene

Bacterial Terminator sequence

Human DNA Polymerase binding site
T7 RNA polymerase binding site

Step 3-3: You complete the PCR and clone it into your DNA construct. You sequence the cloned gene and find four changes relative to your normal PAH sequence:

1. A mutation of a Tyr to a Ser that is observed in approximately 10% of human DNA sequences and localizes to the surface of the protein.

2. A mutation of a Leu to an Ala that is a novel mutation and localized to coiled-coil interaction between the tetrameric PAH subunits.

3. A mutation of a proline to an alanine that is a novel mutation and localized to a loop near the active site of the enzyme.

Circle the two out of the three mutations that are MOST likely to be a potential cause of the patient’s illness.

Explain your choice:

4. You know that the pI for this protein is ~ 6.2 based on sequence analysis. You decide to try purifying it using a anion exchange column.
Step 4-1: Choose a suitable buffer from the list below and explain your choice:

a. 30mMNaCl,100mMTrispH7.5
b. 30mMNaCl,100mMMes pH6.5 c. 30 mM NaCl, 100 mM Citrate pH 5.5

Buffer Choice: _________ Explain:

d. 300mMNaCl,100mMTrispH7.5
e. 300mMNaCl,100mMMes pH6.5 f. 300 mM NaCl, 100 mM Citrate pH 5.5

Step 2: You pass the E.coli lysate after expression over the anion exchange column and test the eluted fractions for the presence of iron. Finding no iron-containing fractions, you analyze the results by SDS-PAGE. Here is what you observe:

Circle yes or no to answer :

Was the PAH:

Expressed?

The correct size?

Soluble?

Bound by the column?

Present in the fractions?

Yes No

Yes No

Yes No

Yes No

Yes No

State a hypothesis that explains the observations above:

Briefly describe one experiment to test your hypothesis:

5. In order to determine which of the two mutations you identified in step 3-3 is responsible for the symptoms in the patient, what biochemical analyses would you perform using your purified protein? Briefly explain, stating the objective of the analyses, the technique/method you would use, the controls you would need and what outcome you would expect for each possibility.

Objective:

Methods/Techniques used:

Outcome/results expected (for each possibility):

6. The effects of glucagon and insulin on the patient’s PAH activity were investigated. The results are shown in the figure below. In addition, the amount of radioactively-labeled phosphate incorporated into PAH with glucagon treatment was found to be nearly seven-fold greater than in controls.

1. How would you interpret these data?

2. Which hormone activates PAH, and why?

For the wild-type allele:

5’-ATG GAC TGG GTA ACT GAT ATG ATA GTG CCC GGG GAA CGC GCG CGG TAA-3’

1. Write the amino acid sequence of the wild-type allele (three letter or single-letter amino acid abbreviation ok).

2. Write the amino acid sequence of the following mutants:

Mutant 1: transition at nucleotide 24

Mutant 2: transition at nucleotide 15

Mutant 3: A C-->A transversion at nucleotide 29

Mutant 4: transition at nucleotide 8

Mutant 5: an insertion of T after nucleotide 14

Mutant 6: a deletion of nucleotide 19

Mutant 7: An insertion of GG after nucleotide 40

3. For each mutant, state whether the mutation is missense, nonsense, frameshift, or silent (it is possible that more than one can apply to the mutation)

You are studying a gene that encodes a particular protein; part of the amino acid sequence of that protein is shown below:

…-His-Val-Pro-Thr-Asp-Leu-Glu-…

You isolate a mutant version of this protein; the mutation abolishes the function of the protein. When you sequence the mutant protein,

you see the following amino acid sequence:

…-His-Val-Leu-Asp-Arg-Leu-Gly-…

Answer/do the following (refer to the Codon Chart below):

a. What was the most likely type of mutation (missense, nonsense, or frameshift) that occurred in the gene
encoding this protein that resulted in the mutated amino acid sequence above? (I am looking for the
simplest, most plausible explanation.)

b. In which codon did the mutation occur? What change actually happened?

c. Write out plausible RNA sequences that encode both the wild-type and mutant proteins

This is the general equation for cellular respiration: C6H12O6 + 6 O2 ? 6 CO2 + 6 H2O + Energy. Carbon dioxide (CO2) is released during which of the following stages of cellular respiration? (choose all that apply)

Discuss the regulation of glycolysis. In your answer ensure you list the key reactions, clearly highlighting the metabolic intermediates, enzymes and hormones involved in the pathway and its regulation. You should also highlight where the regulation differs between muscle and liver cells 20 Marks