Exercises

1. Can you list 10 recombinant proteins which are used in medical practice? Find out where they are used as therapeutics (use the internet).

1. Insulin: Diabetes mellitus.
2. Growth Hormone: Pituitary dwarfism.
3. Blood Clotting Factor VIII: Haemophilia A.
4. Blood Clotting Factor IX: Haemophilia B.
5. Erythropoietin: Anaemia (especially in kidney failure).
6. Interferons (alpha, beta, gamma): Viral infections and cancer.
7. Tissue Plasminogen Activator (tPA): Myocardial infarction (clot buster).
8. Hepatitis B Vaccine: Immunisation against HBV.
9. Follicle Stimulating Hormone (FSH): Infertility treatment.
10. Granulocyte-Colony Stimulating Factor (G-CSF): Neutropenia (during chemotherapy).

2. Make a chart showing a restriction enzyme, the substrate DNA, the cutting site and the product.

• Enzyme: EcoRI
• Recognition Sequence (Substrate):

  5' - G A A T T C - 3'
  3' - C T T A A G - 5'

• Cutting Site: Between G and A on both strands.
• Product (Sticky Ends):

  5' - G                 A A T T C - 3'
  3' - C T T A A                 G - 5'

3. From what you have learnt, can you tell whether enzymes are bigger or DNA is bigger in molecular size? How did you know?

• DNA is much bigger.
• DNA is a long polymer consisting of millions of base pairs (macromolecule). Enzymes are proteins, and even large proteins are coded by only a small segment of the DNA (a gene). In gel electrophoresis, the whole genomic DNA remains at the origin if not fragmented, while enzymes are much smaller units.

4. What would be the molar concentration of human DNA in a human cell? Consult your teacher.

• (Calculated as the number of moles per unit volume of the nucleus. Given 6.6 x 10^9 bp per diploid cell, the concentration is extremely low in molar terms but very high in terms of density within the nucleus.)

5. Do eukaryotic cells have restriction endonucleases? Justify your answer.

• No. Restriction endonucleases are part of the defense mechanism of prokaryotes (bacteria) against viral (bacteriophage) infections. Eukaryotes have other defense mechanisms. If eukaryotes had these enzymes, they would risk cutting their own genomic DNA unless it was highly modified (methylated).

6. Besides better aeration and mixing properties, what other advantages do stirred tank bioreactors have over shake flasks?

• Possess a control system for temperature and pH.
• Equipped with a foam control system.
• Allow for periodic withdrawal of small volumes of culture (sampling ports).
• Can process much larger volumes (100–1000 litres).

7. Collect 5 examples of palindromic DNA sequences.

1. EcoRI: 5'-GAATTC-3'
2. HindIII: 5'-AAGCTT-3'
3. BamHI: 5'-GGATCC-3'
4. PstI: 5'-CTGCAG-3'
5. SalI: 5'-GTCGAC-3'

8. Can you recall meiosis and indicate at what stage a recombinant DNA is made?

• In nature, recombination occurs during the Pachytene stage of Prophase I of Meiosis, where crossing over takes place between non-sister chromatids of homologous chromosomes.

9. Can you think and answer how a reporter enzyme can be used to monitor transformation?

• A reporter gene (e.g., lacZ) codes for an enzyme (β-galactosidase) that produces a visible change (like a blue colour) in the presence of a substrate. If a foreign gene is inserted into the reporter gene, the enzyme is no longer produced (insertional inactivation), and the transformants appear as white colonies, allowing for easy visual screening.

10. Describe briefly the following:

• (a) Origin of replication (ori): A specific DNA sequence where replication starts. It also controls the copy number of the linked DNA.
• (b) Bioreactors: Large vessels where raw materials are biologically converted into specific products using microbes, plant, or animal cells under optimal conditions.
• (c) Downstream processing: The stages of separation and purification of a biosynthetic product before it is ready for marketing.

11. Explain briefly:

• (a) PCR: A technique to synthesize multiple copies of a specific DNA segment in vitro using thermostable DNA polymerase and primers.
• (b) Restriction enzymes and DNA: These enzymes 'inspect' DNA and cut it at specific palindromic sequences, acting as molecular scissors.
• (c) Chitinase: An enzyme used to break open fungal cell walls to release DNA.

12. Distinguish between:

• (a) Plasmid DNA and Chromosomal DNA: Plasmids are small, circular, extra-chromosomal, and autonomously replicating; chromosomal DNA is the main, large genomic DNA of the cell.
• (b) RNA and DNA: RNA has ribose sugar and Uracil; DNA has deoxyribose sugar and Thymine.
• (c) Exonuclease and Endonuclease: Exonucleases remove nucleotides from the ends of DNA; endonucleases cut DNA at specific internal positions.