NCERT Solutions Class 12 Biology Chapter 11

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

Answer:

10 recombinant proteins used in medical practice along with their therapeutic value are as follows:

Q.2 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:

Q.3 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 bigger in the molecular size as compared to enzymes. Because, DNA contains the entire genetic information which is first transcribed into RNA by transcription and then translated into proteins by the process of translation with the help of mRNA, tRNA and ribosomes. A macromolecule DNA has many genes which code for numerous proteins while on the other hand, enzymes are protein molecules which are coded by genes – a small stretch of genomic DNA molecule that can be transcribed into RNA.

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

Answer:

Molar concentration is the concentration measured by the number of moles of solute per litre of solution. The number of moles of human DNA in a human diploid cell will be calculated as follows:

*Total number of chromosomes × 6.023 × 10²³

= 46 × 6.023 × 10­­­²³

=2.77 ×10²⁵ Moles

However, since the volume of solute in each cell varies depending upon the shape, size and type, one cannot calculate the molar concentration of DNA in each human cell.

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

Answer:

No, eukaryotic cells do not have restriction endonucleases.

Justification: Restriction endonucleases are enzymes present in bacteria that restrict the growth of bacteriophages (viruses that infect the bacteria) by cutting DNA at or near a specific recognition nucleotide sequence called restriction site. They are the self-defence mechanisms of the bacteria against the invading virus. The bacterial host DNA is protected from the action of these restriction endonucleases because of certain modifications like methylations present on its host DNA.

The eukaryotic cells have another class of endonucleases that are involved in the cutting of the DNA backbone. They are not called restriction endonucleases. One example is topoisomerase that helps in releasing the tension of supercoiled DNA structure at the time of replication.

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

Answer:

To produce recombinant proteins, bacterial cultures carrying the recombinant gene expressing the desired protein are grown in lab or at industrial scale. Small volumes of culture can be grown in shake flask. However, to produce large quantities, stirred flask bioreactors are used. Besides having better aeration and mixing properties, the other advantages of stirred tank bioreactors over shake flasks are:

1. Air or oxygen can be bubbled in a controlled manner through the reactor.
2. Foam control system can break excess foam produced during stirring.
3. Temperature control system can help control the temperature to the desired levels.
4. pH control system helps control the pH to the desired levels.
5. Sampling ports of the bioreactor help take small volumes of the sample to test at periodic intervals.

Q.7 Write 5 examples of palindromic DNA sequence by following base pair rules. Better try to create a palindromic sequence by following base-pair rules.

Answer:

A palindromic sequence is a sequence of DNA that is same when read from 5′ to 3′ end on one strand 3′ or to 5′ on the complementary strand. These are recognised by restriction endonucleases and are sites for their enzymatic activity, thus breaking the DNA right at that site. Some examples of palindromic DNA sequences are as follows:

• EcoRI : 5’ – G A A T T C – 3’

          3’ – C T T A A G – 5’

• BamHI: 5’ – G G A T C C – 3’

           3’ – C C T A G G– 5’

• HindIII: 5’ – A A G C T T – 3’

           3’ – T T C G A A– 5’

• PstI: 5’ – C T G C A G – 3’

           3’ – G A C G T C – 5’

• EcoRV: 5’ – G A T A T C – 3’

           3’ – C T A T A G – 5’

• Self-designed palindromic sequence: 5’ – C C A T G G– 3’

           3’ – G G T A C C– 5’

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

Answer:

Meiosis is a type of cell division necessary for sexual reproduction in eukaryotes. The number of sets of chromosomes is reduced to half of the original number at the end of meiosis. Meiotic division occurs in two stages, meiosis I and meiosis II. During the first stage, in a diploid cell, homologous chromosomes pair up and exchange genetic material with each other in a process called crossing over. The crossing over happens during the pachytene stage of Prophase I of Meiosis I. Thus, it is at the pachytene stage when the recombination occurs.

Q.9 Can you think about how a reporter enzyme can be used to monitor transformation of host cells by foreign DNA in addition to their role as a selectable marker?

Answer:

A reporter gene is a gene that can be attached to a regulatory sequence of another gene of interest. Genes were chosen as reporter genes confer certain easily identifiable or measurable properties in organisms expressing them. They give an indication of whether a certain gene has been taken up by or expressed in the cell or not and therefore they act as a selectable marker.

β-galactosidase gene is one such reporter gene used widely as a selectable marker. The recombinant DNA carrying the gene of interest is inserted within the coding sequence of the β-galactosidase gene present in the host cell, resulting in the inactivation of the enzyme (insertional inactivation). Thus, in the presence of chromogenic substrate, the transformed cells, which have received the gene of interest, do not produce any colour and give white colonies since the β-galactosidase gene has been inactivated. However, the cells, that did not receive the gene of interest, still carry a functional copy of the β-galactosidase gene, thus producing functional enzyme and give blue colour in the presence of chromogenic substrate. This way, recombinants can be distinguished from non-recombinants on the basis of their ability to produce colour in the presence of chromogenic substrate.

Q.10 Describe briefly the following:

1. Origin of replication
2. Bioreactors
3. Downstream processing

Answer:

1. Origin of replication: The origin of replication is a specific sequence in a genome at which replication or duplication of DNA is initiated. DNA replication may proceed from this point bidirectionally or unidirectionally. The specific structure of the origin of replication varies from species to species, but all share some common characteristics. The pre-replication complex binds at the replication origin and initiates the process of replication by unwinding the DNA. The origin of replication sequences have been exploited by scientists in the process of cloning by linking any foreign DNA with the origin of replication, thus this foreign DNA can replicate and multiply itself in the host organism.
2. Bioreactors: One of the major applications of genetic engineering is the production of recombinant proteins. These are proteins that are produced in the heterologous host after cloning the gene of interest in a suitable vector and expressing the protein under the right conditions in a new host. These host cells can be grown at a small scale in the laboratory in shake flasks but such small volumes cannot yield a good amount of protein product. To produce such proteins in large amounts, bioreactors have been developed where large volumes (100-1000 litres) of culture can be processed under desired and controlled conditions. These are the vessels where the added raw material is converted into specific products using microbial, plant, animal or human cells. It provides the optimal conditions for achieving the desired product by providing optimal growth conditions (temperature, pH, substrate, salts, vitamins, oxygen). A stirred tank bioreactor is the most common kind of bioreactor which provides very good aeration and mixing of all the constituents.
3. Downstream processing: Downstream processing is the recovery and purification of biosynthetic products from natural sources such as animal or plant tissue or fermentation broth. It also includes the recycling of components that can be used again and, the proper treatment and disposal of waste. After completion of a biosynthetic stage, the recombinant product needs to be subjected to a series of processes before it can be marketed as a finished product. These include separation and purification of the product from the bioreactor. The product is mixed with various other things depending upon the nature of the product e.g. preservatives. Such mixed formulations are subjected to clinical trials in case of drugs. Strict quality control testing is required for each product. This downstream processing and quality control testing vary from product to product.

Q.11 Explain briefly:

1. PCR
2. Restriction enzymes and DNA
3. Chitinase

Answer:

1. PCR: PCR (Polymerase Chain Reaction) is a revolutionary method developed by Kary Mullis in the 1980s. It is based on the ability of enzyme DNA polymerase to synthesise new strand of DNA complementary to the template strand. Since DNA polymerase can add a nucleotide only onto a preexisting 3′-OH group, it needs a primer (a small stretch of DNA) to which it can add the first nucleotide. This requirement makes it possible to designate a specific region of template sequence that the scientist wants to amplify. At the end of the PCR reaction, billions of copies of the specific sequence are obtained. PCR reaction requires a DNA template, DNA polymerase, primers and dNTPs (deoxynucleotide triphosphate). It consists of the following steps which are repeated for about 30-40 cycles:

• Denaturation: Carried out at 94°C to denature the DNA.
• Annealing: To allow the primer DNA to bind to the denatured DNA as per the base-pairing rule.
• Extension: Carried out at 72°C where free dNTPs are sequentially added to the growing chain using the template DNA sequence.

Taq polymerase derived from Thermas aquaticus is a commonly used DNA polymerase, in PCR reactions because it is highly thermostable. It can tolerate high temperatures like 94°C during the denaturation step.

2. Restriction enzymes and DNA: Restriction enzymes are enzymes present in bacteria that inhibit the growth of bacteriophages inside the host bacteria and thus, act as a self-defence machinery of the bacteria. They recognise specific sequences in the DNA, called restriction sequence and cut the DNA within or around it depending upon the nature of the restriction enzyme. They inspect the length of the DNA sequence and once the restriction sequence is found, they bind to the DNA and cut each of the two strands of the double helix at a specific point in the sugar phosphate backbone.

The convention for naming these enzymes is as follows: the first letter of the name comes from the genus and the second two letters from the species of the prokaryotic cell from which they were isolated, e.g. EcoRI comes from Escherichia coli RY 13. In EcoRI, R is derived from the name of the strain. Roman numbers following the name indicate the order in which the enzymes were isolated from that strain of bacteria.

Restriction enzymes are extensively used in genetic engineering to form recombinant molecules of DNA, which are composed of DNA from different sources/genomes.

3. Chitinase: Chitinases are digestive enzymes that break down glycosidic bonds in chitin. Chitin composes the cell walls of fungi and exoskeletal elements of some animals (including worms and arthropods). Chitinase is usually found in these organisms as it helps in reshaping their own cell wall. However, chitinase has been exploited by genetic engineering scientists and is used widely to digest fungal cell walls during DNA isolation.

Q.12 Discuss with your teacher and find out how to distinguish between

1. Plasmid DNA and Chromosomal DNA
2. RNA and DNA
3. Exonuclease and Endonuclease

Answer:

a. Plasmid DNA and Chromosomal DNA

b. RNA and DNA

c. Exonuclease and Endonuclease