Biology Test - 8

Key Points

• Thermus aquaticus is a bacterium that thrives in extreme heat conditions like hot springs.
• It is used to obtain an enzyme called Taq polymerase.
• This enzyme remains active even in high temperatures.
• Hence, it is a thermostable DNA polymerase that can be used for repeated amplification of DNA in Polymerase Chain Reaction (PCR).

Important Points

• PCR is the process of amplifying or making multiple copies of a desired fragment of DNA.
• The process involves 3 basic steps:

1. Denaturation -
   • It is the process by which 2 strands of ds-DNA separates to form 2 single strands.
   • This is achieved by applying heat that helps in breaking the H-bonds between the 2 DNA strands.
2. Annealing -
   • In this process, 2 sets of primers bind to specific regions on the separated strands of DNA.
   • Primers - are small, chemically synthesized oligonucleotides that are complimentary to specific regions of DNA.
3. Extension -
   • This step involves extension of the primers using the thermostable DNA polymerase (Taq polymerase) in the presence of deoxynucleotides.

• These steps are repeated in cycles such that we get about a billion copies of the DNA in 30 cycles.

Additional Information

• Bt-toxin -
  • It is obtained from the bacterium Bacillus thuringiensis.
  • It is toxic for certain insects that attack plants.
  • The gene for this toxin is called cry gene, which can be genetically introduced into a host plant to get resistance from a particular insect pest.
• Ti plasmid -
  • ​It is obtained from Agrobacterium tumifaciens.
  • This plasmid can be genetically modified to be used as cloning vectors.
  • It can efficiently deliver the gene of our interest into the host plant.
• pBR322 -
  • ​It is a plasmid that is widely used as E. coli cloning vector.
  • It contains ampicillin and tetracycline resistance genes as selectable markers.

Key Points

• Genetic engineering became possible with the discovery of certain enzymes that could provide specific desired fragments of DNA.
• These are called restriction endonucleases.
• Nucleases are a group of enzymes that can cut polynucleotides or DNA.
• These nucleases can be:
  • Exonuclease - These can cut DNA only from the ends (5' or 3'-end).
  • Endonuclease - These can cut within a DNA sequence.
• The endonucleases that have the ability to cut at a specific site within DNA are known as restriction endonucleases or simply, restriction enzymes.
• Examples - EcoRI, Hind III.
• The sites at which they cut are called restriction sites.
• These enzymes also have a specific recognition sequence which is a palindromic nucleotide sequence.
• These sequences are base pair sequences that read the same on both the strands of DNA when orientation of reading is same, i.e., either 5' to 3' or 3' to 5'.
• The action of restriction endonucleases on DNA is known as restriction digestion as it cleaves the DNA.

Important Points

• Restriction endonucleases cut at the same points on both the strands.
• They may produce 2 types of fragment ends:
  • Sticky/Cohesive End - It is produced by offset cuts of DNA i.e., not in the middle of the recognition sequence.
  • Blunt End - It is produced when the cut is exactly at the middle of the palindrome sequence.

Explanation:

• Cohesive ends have an overhanging single-stranded end of DNA.
• This overhanging end can form H-bonds with its complementary sequence, if available.
• This property is utilized for joining 2 different DNA fragments with same overhanging ends.
• Hence, they are termed as sticky or cohesive ends.

Concept-

• DNA is a long polymer of deoxyribonucleotides.
• The length of DNA is usually defined as a number of nucleotides (or a pair of nucleotides referred to as base pairs) present in it.
• This also is the characteristic of an organism.
• An alien piece of DNA has become part of a chromosome, which has the ability to replicate.
• In a chromosome, there is a specific DNA sequence called the origin of replication, which is responsible for initiating replication.
• Therefore, for the multiplication of any alien piece of DNA in an organism, it needs to be a part of a chromosome(s) that has a specific sequence known as ‘origin of replication.
• Thus, alien DNA is linked with the origin of replication, so that, this alien piece of DNA can replicate and multiply itself in the host organism.
• This can also be called cloning or making multiple identical copies of any template DNA.

Key Points

Methylases are not required for DNA cloning.

• Methylases add methyl groups (—CH3) to adenine or cytosine bases within the recognition sequence, which is thus modified and protected from the endonuclease.

Additional Information

DNA ligase

• DNA ligase is also called molecular glue.
• This enzyme joins DNA fragments together by forming phosphodiester bonds between nucleotides.

A vector

• Plasmid (autonomously replicating circular extra-chromosomal DNA).
• These plasmid DNA act as vectors to transfer the piece of DNA attached to it. 

Restriction endonucleases

• In the year 1963, the two enzymes responsible for restricting the growth of bacteriophage in Escherichia coli were isolated.
• One of these added methyl groups to DNA, while the other cut DNA. The later was called restriction endonuclease.

Key Points

• ADA stands for adenosine deaminase, an enzyme crucial for the immune system.
• The deficiency of ADA is a severe combined immunodeficiency (SCID) disorder.
• It is caused by the deletion of the gene encoding ADA.
• If not treated properly, the disease can even be fatal because of the weak immune system.

Important Points

• Enzyme Replacement Therapy (ERT) -
  • Healthy enzymes are injected into the patient's body.
  • It is not a permanent cure but reduces infections by strengthening the immune system.
  • Repeated injections are required.
  • Functional ADA can be injected by this process.
• Gene Therapy -
  • The first clinical gene therapy was done on a ADA deficient patient.
  • In this process, lymphocytes from the patient are grown in culture.
  • Then cDNA of functional ADA is introduced into the lymphocytes with the help of retroviral vectors.
  • These genetically modified lymphocytes are then infused into the patient.
  • Periodic infusion is also required in this process.
• Radiation Therapy -
  • It is a type of cancer treatment where high doses of radiation are used to kill the cancer cells.
  • Thus, this treatment method is NOT suitable for treating ADA deficiency.
• Hematopoietic Stem Cell Transplantation (HSCT) -
  • Hematopoietic stem cells (HSC) are immature cells present in the bone marrow that can develop into any type of blood cell.
  • In this process of treatment, healthy HSCs that can produce ADA are obtained from the bone marrow and introduced into the patient.
  • This helps in rebuilding the immune system.
  • The results are better when done in infant stage and the donor is a close relative.
• Therefore from the given options, only radiation therapy is not a possible treatment method for ADA deficiency.

Key Points

• EcoRI is a restriction enzyme.
• Restriction enzymes or restriction endonucleases are endonucleases that have the ability to cut at a specific site within DNA.
• Restriction enzymes were first isolated in 1963 as an enzyme that restricted growth of bacteriophage in E. coli.
• These enzymes are named in a specific manner.
• For example, the name 'EcoRI' has been given on the basis of the following:
  • The first letter "E" comes from the initial of the genus from which it is isolated i.e., Escherichia.
  • The second two letters "co" refers to the species from which it is isolated i.e., Escherichia coli.
  • The letter "R" is derived from the strain of bacteria from which it is isolated i.e., RY13.
  • The last roman numeral "I" denotes the order in which the enzymes were isolated from that strain.

Therefore, "co" in EcoRI stands for 'coli'.

Additional Information

• Restriction enzymes have a specific recognition sequence which is a palindromic nucleotide sequence.
• These sequences are base pair sequences that read the same on both the strands of DNA when orientation of reading is same, i.e., either 5' to 3' or 3' to 5'.
• The sites at which they cut are called restriction sites.
• The action of restriction endonucleases on DNA is known as restriction digestion as it cleaves the DNA.
• Restriction endonucleases cut at the same points on both the strands.
• They may produce 2 types of fragment ends:
  • Sticky/Cohesive End - It is produced by offset cuts of DNA i.e., not in the middle of the recognition sequence.
  • Blunt End - It is produced when the cut is exactly at the middle of the palindrome sequence.
• Restriction enzymes create same cohesive ends on different DNA fragments.
• These 2 cohesive ends can then be joined together by ligase enzyme.
• It produces a recombinant DNA molecule, which can then be inserted into a host cell for cloning.

Key Points

• Isolation of DNA from a cell is the first step for any genetic engineering process.
• Restriction enzymes can work only when the DNA molecule is in the pure form, free from other macromolecules.
• Thus DNA needs to be isolated from a cell before proceeding with restriction digestion.

Steps of Isolation of DNA:

1. Treatment with Digestive Enzymes -
   • Digestive enzymes help to break down the cell wall of bacterial, plant or fungal cell.
   • Lysozyme- digests the bacterial cell wall.
   • Cellulase- digest the plant cell wall.
   • Chitinase- digest the fungal cell wall.
2. Removing other macromolecules -
   • RNA and proteins are removed by this process.
   • Protease- cleaves protein molecules.
   • Ribonuclease- removes the RNA.
3. Precipitation of purified DNA -
   • This is achieved by the addition of chilled ethanol.
   • The charged DNA molecules move away from ethanol and precipitate.
4. Removal of DNA -
   • Fine threads of purified DNA can be observed in the suspension.
   • This can be removed by a process called spooling.
   • Spooling is the process that helps to scoop up long DNA threads by swirling it around a glass rod or spooling stick.

Important Points

• Deoxyribonuclease or DNase is the enzyme that can break up DNA molecules.
• We need an intact DNA molecule to digest it with desired restriction enzymes.
• If DNA molecules are damaged while isolating it from the cell, it would not solve the purpose.

Thus, deoxyribonuclease is an enzyme that is not used while isolation of DNA.

Key Points

• 'Cry' protein is obtained from the bacterium Bacillus thuringiensis.
• It is also known as the Bt-toxin.
• This toxin is effective in killing insects like -
  • Lepidopterans - Tobacco budworm, armyworm.
  • Coleopterans - Beetles.
  • Dipterans - Flies, mosquitoes.
• The gene encoding this toxin is named as 'cry' gene.
• These genes produce insect-group specific toxins and thus can be introduced into crop plant genomes to make them pest-resistant.
• The genes cryIAc and cryIIAb are effective against cotton bollworms, while the gene cryIAb is effective against corn borers.
• Bt-cotton is a genetically modified crop plant that carries cry genes to make it resistant against cotton bollworms.

Mechanism of Action of Cry protein:

• The bacteria produce crystals containing insecticidal Cry proteins during a particular growth phase.
• The protein exists in the inactive protoxin form in the bacteria.
• It gets activated in the alkaline gut of insects, where the crystals get solubilized.
• This releases the active toxin, which binds to the midgut epithelial cells of insects.
• This creates pores causing cell swelling and lysis.
• This leads to the death of the insect.

Additional Information

• Agrobacterium tumifaciens - 
  • This is a bacterium that contains the Ti-plasmid.
  • This plasmid can be genetically modified to be used as cloning vectors.
  • It can efficiently deliver the gene of our interest into the host plant.
• Thermus aquaticus -
  • It is a bacterium that thrives in extreme heat conditions like hot springs.
  • It is used to obtain an enzyme called Taq polymerase.
  • This enzyme remains active even in high temperatures and hence used in PCR.
• Escherichia coli - 
  • It is widely used in biotechnological processes for various purposes.
  • EcoRI is a restriction enzyme obtained from this bacteria.
  • The cloning vector pBR322 is also obtained from E. coli.

Concept:

• Genetic engineering is important for producing more effective and safer therapeutic drugs.
• Recombinant therapeutics are advantageous because they do not induce any unwanted immunological responses.
• Diabetes is one such disease where the patients need to take insulin at regular intervals.
• This necessitates use of insulin from non-human sources.
• Insulin was previously extracted from the pancreas of slaughtered cattle and pigs.
• But this caused allergies and other reactions in the patients.
• This problem was solved by producing human insulin by genetic engineering using bacteria.

Important Points

• Human Insulin:
  • Human insulin consists of 2 short peptide chains - peptide A and B.
  • These are linked to each other by disulphide bridges.
  • Insulin is produced as proinsulin, which is a prohormone.
  • Proinsulin contains an extra C-peptide that needs to be cleaved during maturation of proinsulin to insulin.
• Eli Lilly was an American company who produced human insulin using genetic engineering in 1983 using the bacterium Escherichia coli.
• They prepared 2 DNA sequences corresponding to the A and B peptides.
• These DNA sequences were then inserted into E. coli plasmids.
• The A and B peptides were produced separately and extracted.
• These peptide chains were then linked by forming disulphide bonds between them.
• This produced the human insulin.

Concept:

• Gene therapy refers to a collection of methods involving genetic engineering techniques that allows correction of a gene defect by alteration or replacement.
• Correction of a gene defect involves the delivery of a normal gene that could compensate for the non-functional gene.
• This is achieved by inserting the genes into cells using retroviral vectors.

Important Points

• The first clinical gene therapy was done in 1990 to a 4 years old girl for the treatment of ADA deficiency.
• ADA stands for adenosine deaminase, an enzyme crucial for the immune system.
• The deficiency of ADA is a severe combined immunodeficiency (SCID) disorder.
• It is caused by the deletion of the gene encoding ADA.
• If not treated properly, the disease can even be fatal because of the weak immune system.

Explanation:

• ADA is mostly produced in the lymphocytes and thus lymphocytes are used gene therapy technique.
• The lymphocytes are first obtained from the patient blood and then grown in culture medium in vitro.
• A cDNA (complementary DNA) for functional ADA is introduced into the lymphocytes.
• These genetically engineered lymphocytes are then returned to the patient.
• These lymphocytes, having the functional ADA gene, produce ADA in the patient.
• But the patient would still require periodic infusion of such genetically engineered lymphocytes.
• Permanent cure by gene therapy can be obtained if a functional gene is isolated from bone marrow cells and then introduced into defective cells at an early embryonic stage.

The correct answer is A transgenic cow.

Key Points

• Organisms that have altered genomes through artificial means are known as transgenic.
• The first transgenic cow was Rosie and was produced in 1997.
  • It consists of the human alpha-lactalbumin gene.
  • Its milk contained 2.4gms of human α-lactalbumin per litre of milk.
  • It produces human protein-enriched milk, which was a nutritionally more balanced product for human babies than natural cow milk as it contains 'human alpha-lactalbumin'.

Additional Information

Reasons for production of transgenic animals:

• Study of Physiology & Development -
  • Study of how genes and their regulation affects the physiological functioning of the body.
  • Complex growth factors like insulin can be studied.
• Study of Diseases -
  • Transgenic animals serve as model organisms for studying human diseases.
  • They help in understanding the disease development process from genes.
  • They are also used for investigating new treatment methods.
  • Transgenic models exist for diseases like cancer, cystic fibrosis, rheumatoid arthritis and Alzheimer's.
• Biological Products -
  • Many biological products that are essential for formulating medicines of use are often expensive.
  • These products can be obtained easily from transgenic animals.
  • This can be achieved by just introducing the gene responsible for the desired product into a suitable organism.
  • Example - Alpha-1 antitrypsin is a human protein that is required for emphysema treatment.
• Vaccine Safety -
  • Transgenic mice can be used to test safety of vaccines before using for humans.
• Toxicity Testing -
  • Transgenic animals can be made more sensitive to certain chemicals or drugs.
  • These animals are exposed to the toxic substances and their effects are then studied.