Can you list 10 recombinant proteins which are used in medical practice? Find out where they are used as therapeutics (use the internet).
|Recombinant proteins||Therapeutic use|
|Human growth hormone||Dwarfism|
|Tissue plasminogen activator||Myocardial infarction|
|Insulin-like growth factor||Childhood growth anabolic effects in adults|
|Erythropoietin||Stimulation of production of erythrocytes|
|Interferon||Treatment of infectious diseases like multiple sclerosis|
|Alpha-galactosidase A||Enzyme replacement therapy|
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.
From what you have learnt, can you tell whether enzymes are bigger or DNA is bigger in molecular size? How did you know?
Solution: In nature 20 different kinds of amino acids are found. Average protein length is around 300 residues of amino acids.
DNA is made up of polymers of nucleotides. DNA polymers can be enormous molecules containing million of nucleotides. Like human chromosome is made up of 220 million base pairs. So, DNA is bigger in molecular size.
Also, DNA contains genetic information for the development and functioning of all living organisms. It contains instructions for the synthesis of proteins and DNA molecules. On the other hand, enzymes are proteins which are synthesised from a small stretch of DNA known as ‘genes’, which are involved in the production of the polypeptide chain.
What would be the molar concentration of human DNA in a human cell? Consult your teacher.
Solution: The molar concentration of human DNA in a human diploid cell is as follows:
⇒ Total number of chromosomes × 6.023 × 1023
⇒ 46 × 6.023 × 1023.
Do eukaryotic cells have restriction endonucleases? Justify your answer.
Solution: No, eukaryotic cells do not have restriction endonucleases. This is because the DNA of eukaryotes is highly methylated by a modification enzyme, called methylase. Methylation protects the DNA from the activity of restriction enzymes. These enzymes are present in prokaryotic cells where they help prevent the invasion of DNA by virus.
Besides better aeration and mixing properties, what other advantages do stirred tank bioreactors have over shake flasks?
Solution: Stirred tank bioreactors provide a better agitation system, oxygen delivery system, foam system, pH control system, temperature control system and provide a better provision for cleaning and sterilization.
Collect 5 examples of palindromic DNA sequences by consulting your teacher. Better try to create a palindromic sequence by following base-pair rules.
Solution: The palindromic sequence is a certain sequence of the DNA that reads the same whether read from 5’ → 3’ direction or from 3’→ 5’direction. They are the site for the action of restriction enzymes. Most restriction enzymes are palindromic sequences.
Five examples of palindromic sequences are:
Can you recall meiosis and indicate at what stage a recombinant DNA is made?
Solution: During the pachytene stage of prophase I, crossing over of chromosomes takes place where the exchange of segments between non-sister chromatids of homlogous chromosomes takes place. This results in the formation of recombinant DNA.
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?
Solution: A reporter gene can be used to monitor the transformation of host cells by foreign DNA. They act as a selectable marker to determine whether the host cell has taken up the foreign DNA or the foreign gene gets expressed in the cell. The researchers place the reporter gene and the foreign gene in the same DNA construct. Then, this combined DNA construct is inserted in the cell. Here, the reporter gene is used as a selectable marker to find out the successful uptake of genes of interest (foreign genes). An example of reporter genes includes lac Z gene, which encodes a green fluorescent protein in a jelly fish.
Describe briefly the following:
(a) Origin of replication (b) Bioreactors
(c) Downstream processing
- Origin of replication − Origin of replication is defined as the DNA sequence in a genome from where replication initiates. The initiation of replication can be either uni-directional or bi-directional. A protein complex recognizes the ‘on’ site, unwinds the two strands, and initiates the copying of the DNA.
- Bioreactors − Bioreactors are large vessels used for the large-scale production of biotechnology products from raw materials. They provide optimal conditions to obtain the desired product by providing the optimum temperature, pH, vitamin, oxygen, etc. Bioreactors have an oxygen delivery system, a foam control system, a pH, a temperature control system, and a sampling port to obtain a small volume of culture for sampling.
- Downstream processing − Downstream processing is a method of separation and purification of foreign gene products after the completion of the biosynthetic stage. The product is subjected to various processes in order to separate and purify the product. After downstream processing, the product is formulated and is passed through various clinical trials for quality control and other tests.
(b) Restriction enzymes and DNA
- PCR: In PCR reaction multiple copies of gene of interest is synthesised in vitro using two sets of primers and the enzyme DNA polymerase. The enzyme extends the primers using the nucleotides provided in the reaction and the genome DNA as template. The segment of DNA can be amplified to approximately billion times.
- Restriction enzymes and DNA: Restriction enzymes are also called molecular scissors. By the use of restriction enzymes it is possible to cut DNA sequence. It acts on both the strands and produce a break. The cut can be overhanging called sticky ends. Sticky ends can make H-bonds with their complementary cut counterparts by DNA ligase.
- Chitinase: Chitinase is a class of enzymes used for the degradation of chitin, which forms a major component of the fungal cell wall. Therefore, to isolate the DNA enclosed within the cell membrane of the fungus, enzyme chitinase is used to break the cell for releasing its genetic material.
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
|Plasmid DNA||Chromosomal DNA|
|It carries genes for sexuality, antibiotic resistance but not any vital genes||It carries vital genes|
|It can replicate independently without main genome||It can’t replicate without main genome|
|Cells can survive without them||Cells can’t survive without them|
|It is single stranded||It is double stranded|
|It contains ribose sugar||It contains deoxyribose sugar|
|It is not the genetic material in humans||It is the genetic material in humans|
|Uracil is present in place of thymine||Thymine is present in place of uracil|
|It removes nucleotides from the ends of DNA||It makes cut at a specific position within the DNA|
– The End –