Exercises
Can you list 10 recombinant proteins which are used in medical practice? Find out where they are used as therapeutics (use the internet).
Answer
(1) Human insulin (Humulin) – diabetes mellitus.
(2) Human growth hormone (somatotropin) – growth hormone deficiency, dwarfism.
(3) Interferon-α – hepatitis B and C, some cancers (e.g. hairy cell leukaemia).
(4) Interferon-β – multiple sclerosis.
(5) Tissue plasminogen activator (tPA) – dissolving blood clots in heart attack and stroke.
(6) Erythropoietin (EPO) – anaemia due to chronic kidney failure.
(7) Blood clotting factor VIII – haemophilia A (factor IX for haemophilia B).
(8) Hepatitis B surface antigen – recombinant hepatitis B vaccine.
(9) Interleukin-2 – stimulating immune response, some kidney cancers.
(10) Granulocyte colony stimulating factor (G-CSF) – raising white blood cell count after chemotherapy.
Others: α-1 antitrypsin (emphysema), recombinant streptokinase (clot buster), DNase (cystic fibrosis), monoclonal antibodies.
Make a chart (with diagrammatic representation) showing a restriction enzyme, the substrate DNA on which it acts, the site at which it cuts DNA and the product it produces.
Answer
Restriction enzyme: EcoRI (from Escherichia coli RY 13).
Substrate DNA: double-stranded DNA carrying the palindromic recognition sequence
5′ — G A A T T C — 3′
3′ — C T T A A G — 5′
Site of cutting: between G and A on both strands (in the sugar-phosphate backbone), i.e. 5′ — G | AATTC — 3′ on the upper strand and 3′ — CTTAA | G — 5′ on the lower strand. The cuts are a little away from the centre of the palindrome but between the same two bases on the two strands.
Product: two DNA fragments, each with a single-stranded overhang (sticky end):
Fragment 1: 5′ — G 3′ / 3′ — CTTAA 5′ and Fragment 2: 5′ AATTC — 3′ / 3′ G — 5′
The AATT overhangs are complementary, so fragments cut by EcoRI (from any source) can pair by hydrogen bonds and be joined by DNA ligase to form recombinant DNA.
From what you have learnt, can you tell whether enzymes are bigger or DNA is bigger in molecular size? How did you know?
Answer
DNA is much bigger. A DNA molecule contains millions of nucleotides (human DNA has about 3.3 × 10⁹ bp per haploid set, and each chromosome's DNA is centimetres long), whereas an enzyme is a protein of usually a few hundred to a few thousand amino acids. Evidence: a restriction enzyme moves along ("inspects") the length of the DNA to find its recognition sequence; DNA is so long that it is seen as fine threads after ethanol precipitation and can be spooled; and DNA has to be cut into fragments before it can be separated by electrophoresis.
What would be the molar concentration of human DNA in a human cell? Consult your teacher.
Answer
Given: a diploid human cell contains about 6.6 × 10⁹ bp of DNA; Avogadro's number = 6.022 × 10²³.
Moles of base pairs per cell = 6.6 × 10⁹ ÷ 6.022 × 10²³
⇒ ≈ 1.1 × 10⁻¹⁴ mol.
So a human cell contains about 1.1 × 10⁻¹⁴ moles of DNA base pairs. If this is taken as present in the nucleus (volume of a nucleus about 6 µm across ≈ 1.1 × 10⁻¹³ L), the concentration of base pairs works out to roughly 1.1 × 10⁻¹⁴ ÷ 1.1 × 10⁻¹³ ≈ 0.1 M.
Do eukaryotic cells have restriction endonucleases? Justify your answer.
Answer
No. Restriction endonucleases are found in bacteria (prokaryotes), where they act as a defence system that "restricts" the growth of invading bacteriophages by cutting the foreign DNA; the bacterium protects its own DNA by methylation. All known restriction enzymes have been isolated from bacteria (900+ enzymes from over 230 strains). Eukaryotic cells do not have this restriction–modification system; their DNA is protected in a nucleus and they have other defence mechanisms, though they do have other nucleases for DNA repair and recombination.
Besides better aeration and mixing properties, what other advantages do stirred tank bioreactors have over shake flasks?
Answer
(i) They handle large volumes (100–1000 litres or more) of culture, so products can be made on an industrial scale. (ii) They have a temperature control system and a pH control system to keep conditions optimal. (iii) A foam control system prevents foaming. (iv) Sampling ports allow small volumes to be withdrawn periodically to monitor the culture. (v) Sterile air can be sparged and oxygen supply controlled; conditions can be kept sterile. (vi) Continuous culture is possible, keeping cells in the exponential phase for higher yield.
Collect 5 examples of palindromic DNA sequences by consulting your teacher. Better try to create a palindromic sequence by following base-pair rules.
Answer
In a DNA palindrome, both strands read the same in the 5′
⇒ 3′ direction.
(1) 5′-GAATTC-3′ / 3′-CTTAAG-5′ (EcoRI)
(2) 5′-GGATCC-3′ / 3′-CCTAGG-5′ (BamHI)
(3) 5′-AAGCTT-3′ / 3′-TTCGAA-5′ (HindIII)
(4) 5′-GTCGAC-3′ / 3′-CAGCTG-5′ (SalI)
(5) 5′-CTGCAG-3′ / 3′-GACGTC-5′ (PstI)
Creating one: take any half-sequence, e.g. 5′-ACG, and add its reverse complement CGT
⇒ 5′-ACGCGT-3′; the complementary strand 3′-TGCGCA-5′ also reads ACGCGT from 5′ to 3′.
Can you recall meiosis and indicate at what stage a recombinant DNA is made?
Answer
Naturally recombinant DNA is formed during the pachytene stage of prophase I of meiosis, when crossing over takes place between non-sister chromatids of homologous chromosomes with the help of the enzyme recombinase. The exchanged segments produce new combinations of genes, which are visible as chiasmata in diplotene.
Can you think and answer how a reporter enzyme can be used to monitor transformation of host cells by foreign DNA in addition to a selectable marker?
Answer
A reporter gene codes for an enzyme whose activity can be seen or measured easily, e.g. β-galactosidase (lacZ), luciferase or GFP. The foreign DNA is inserted within the coding sequence of the reporter gene. On a medium with a chromogenic substrate, cells having a plasmid without insert produce active β-galactosidase and form blue colonies, while cells with the recombinant plasmid have the gene inactivated (insertional inactivation) and form colourless colonies. Thus the selectable marker (e.g. ampicillin resistance) picks out transformed cells, and the reporter enzyme further distinguishes recombinants from non-recombinants in a single plate. Alternatively, a reporter gene placed next to the gene of interest shows by its activity (colour or light) that the foreign DNA has entered and is being expressed.
Q10. Describe briefly the following
(a) Origin of replication
(b) Bioreactors
(c) Downstream processing
Ans. (a) Origin of replication (ori): a specific DNA sequence from which replication starts. Any DNA linked to it can replicate in the host cell. It also controls the copy number of the linked DNA, so a vector with a high-copy ori is used to obtain many copies of the target DNA.
(b) Bioreactors: large vessels (100–1000 litres) in which raw materials are biologically converted into specific products using microbial, plant, animal or human cells. They provide optimum temperature, pH, substrate, salts, vitamins and oxygen. The common stirred-tank type has an agitator, oxygen delivery, foam control, temperature and pH control systems and sampling ports.
(c) Downstream processing: the processes after the biosynthetic stage that make the product ready for marketing – separation and purification of the product, formulation with suitable preservatives, clinical trials (for drugs) and strict quality control testing. These steps vary from product to product.
Q11. Explain briefly
(a) PCR
(b) Restriction enzymes and DNA
(c) Chitinase
Ans. (a) PCR (Polymerase Chain Reaction): in vitro amplification of a gene of interest using two primers, deoxynucleotides and a thermostable DNA polymerase (Taq polymerase from Thermus aquaticus). Each cycle has three steps – denaturation of double-stranded DNA by heat, annealing of primers to the single strands, and extension of primers by the polymerase. Repeating the cycle about 30 times produces about a billion copies.
(b) Restriction enzymes and DNA: restriction endonucleases recognise specific palindromic sequences in DNA and cut both strands at specific points in the sugar-phosphate backbone, usually producing sticky ends. DNA from different sources cut with the same enzyme has matching sticky ends and can be joined by DNA ligase to make recombinant DNA. They are therefore called "molecular scissors".
(c) Chitinase: an enzyme that breaks down chitin, the main component of fungal cell walls. It is used to break open fungal cells to release DNA during isolation of genetic material (like lysozyme for bacteria and cellulase for plant cells).
Q12. Discuss with your teacher and find out how to distinguish between
(a) Plasmid DNA and Chromosomal DNA
(b) RNA and DNA
(c) Exonuclease and Endonuclease
Ans. (a) Plasmid DNA is small, circular, extra-chromosomal DNA found in bacteria (and some yeasts), replicates autonomously, may exist in many copies per cell and carries non-essential genes such as antibiotic resistance; it is used as a vector. Chromosomal DNA is large, carries all the essential genes of the organism, is present usually in one (prokaryote) or two (diploid eukaryote) copies, and is linear and associated with histones in eukaryotes.
(b) RNA has ribose sugar and uracil, is usually single-stranded, less stable and acts mainly in protein synthesis (mRNA, tRNA, rRNA), with catalytic roles in some cases. DNA has deoxyribose sugar and thymine, is double-stranded, more stable and is the genetic material of most organisms.
(c) Exonucleases remove nucleotides one by one from the free ends of a DNA strand. Endonucleases make cuts at specific positions within the DNA molecule (restriction endonucleases cut at specific recognition sequences).
Important Extra Questions
Who constructed the first recombinant DNA and how?
Answer
Stanley Cohen and Herbert Boyer (1972). They cut out an antibiotic resistance gene from a plasmid and linked it to a native plasmid of Salmonella typhimurium using restriction enzymes and DNA ligase.
How are DNA fragments separated and visualised?
Answer
By agarose gel electrophoresis: negatively charged DNA moves towards the anode, smaller fragments moving farther. The bands are stained with ethidium bromide and seen as orange bands under UV light; they are then cut out and extracted (elution).
What is insertional inactivation in pBR322?
Answer
If foreign DNA is ligated at the BamHI site in the tetracycline resistance gene, the recombinant loses tetracycline resistance but keeps ampicillin resistance. Recombinants grow on ampicillin medium but not on tetracycline medium.
How is a bacterial cell made competent?
Answer
By treating it with a divalent cation such as calcium, then incubating with recombinant DNA on ice, giving a heat shock at 42 °C and returning it to ice.
Name two vectors used for gene transfer into plants and animals.
Answer
The disarmed Ti plasmid of Agrobacterium tumefaciens for plants, and disarmed retroviruses for animal cells.