BIOTECHNOLOGY AND ITS APPLICATIONS

The applications of biotechnology include:

• Therapeutics
• Diagnostics
• Genetically modified crops for agriculture
• Processed food
• Bioremediation
• Waste treatment
• Energy production

Q1. Which of the following factors contributed to green revolution?

1. Use of improved crop varieties
2. Use of agro chemicals
3. Both a and b
4. Only b

Ans: 3

The second time, food production was increased by organic farming i.e., by the use of manures, biofertilizers, biopesticides, etc.

The third time, or in the recent time, the food production has been increased by the use of genetically engineered plants or GMO or transgenics.

Q2. Which of the following are the advantages of GM plants?

1. GM crops are more tolerant to abiotic stresses (cold, draught, salt, heat)
2. GM technology helps to reduce post harvest losses
3. GM technology has increased efficiency of mineral usage by plants
4. GM technology has enhanced nutritional value of food
5. All of the above

Ans: 5

Q3. Golden rice is genetically modified rice, which is enriched in:

1. Vitamin B
2. Vitamin A
3. Vitamin C
4. Vitamin D

Ans: 2

Q4. Agrobacterium tumifaciens cause crown gall in:

1. Dicot plants
2. Monocot plants
3. Both a and b
4. Gymnosperms

Ans: 1

Q5. Which of the following is true for Bacillus thuringiensis?

1. It is a soil bacterium
2. This bacterium produces a toxin, which is effective against boll worm, beetles, insects and flies
3. The bacterium produces toxin during sporulation in the form of proto-toxin
4. The bacterial toxin is active only under alkaline condition
5. All of the above

Ans: 5

The Bt toxin is effective against lepidopterans (boll worm, army worm); coleopterans (beetles); dipterans (insects, flies). The bacterium releases toxin in an inactive form. However, when the toxin enters into the intestine of the gut – the toxin in an alkaline form becomes toxic. It combines with the cells of the intestine and causes cell lysis leading to the formation of pore in the intestine. This leads to the death of the organism.

The toxix protein is called Cry protein (it is the product of a gene and that’s why the first letter is written in capital letter). However, when we write a gene – all letters should be in small letter like cry gene.

Q6. Which of the following cry genes are responsible for controlling bollworms?

1. cryIAb
2. cryIAc
3. cryIIAb
4. cryIAc and cryIIAb

Ans: 4

Q7. Which of the following cry gene is responsible for controlling corn borer?

1. cryIAb
2. cryIAc
3. cryIIAb
4. cryIAc and cryIIAb

Ans: 1

Biotechnology applications: NEMATODE RESISTANT TRANSGENIC PLANT

Meloidogyne incognita – root-knot nematode (RKN) – also known as cotton root-knot nematode. It is a plant parasite roundworm. This nematode is present in the soil and infects the roots of the plant by cutting roots. Wherever a cut is made by the nematode, that portion gets thickened – this is called root-knot disease. This drastically reduces the yield. Nematode resistant plants have been produced using RNAi – RNA interference technique. This technique is associated with mRNA silencing. In all eukaryotic cells, RNA interference defense mechanism is present.

In RNAi technology, a dsRNA is introduced into the cell. The dsRNA is soon attacked by DICER enzyme that cuts dsRNA into different pieces, called small RNAi. The small RNAi’s get attached to proteins and form RISC (RNA-induced silencing complex). This RISC forms single stranded RNA fragments. Once mRNA from nucleus enters into the cytoplasm – these single stranded RNA fragments join to the mRNA wherever they see complementary sequence. This results in the breakage of mRNA into fragments and thus not viable to form proteins – that’s why it is also called as mRNA silencing. How this technology is used in forming nematode resistant plant?

Nematode specific gene is transferred to the plant cell using the vector Agrobacterium. The nematode specific gene should have both sense and antisense strand and both strands should express. In other words, both the strands of DNA should transcribe and form mRNA. Since both DNA strands are complementary – their mRNA sequence would also be complementary. Once mRNAs are form – they join together and form dsRNA. So, once the nematode infects the plant, it won’t be able to form the protein responsible for the cut in plant root.

Biotechnology applications: PRODUCTION OF TRANSGENIC PRODUCTS

1. Hirudin: it is an anticoagulant, which is obtained from the leech. In order to obtain hirudin from leech – how many leeches would be required? Well, many leeches would be required for this purpose and that’s why, it is a very tedious and expensive procedure. On the other hand, using rDNA technology, we can obtain hirudin in large amount from the plant.

Q. How plant can produce hirudin?

A. Gene responsible for hirudin is synthesized artificially. The gene is then transferred to the Brassica napus plant in such a way that it finds expression in the seeds of it. So, we can obtain pure hirudin from the seeds of this transgenic plant.

2. Production of flavr-savr variety of tomato: when any fruits or vegetables are harvested – they start rotting after some time. This rotting is due to the dissolution of the middle lamella. If this dissolution is reduced then fruits and vegetables could remain fresh for a longer period of time. The enzyme responsible for the dissolution of middle lamella is POLYGALACTOURONASE. If the gene responsible for this enzyme is masked then the dissolution of the middle lamella can be delayed. This gene has been masked in the flavr-savr variety of tomato. This way, the flavor of tomato is retained and the freshness of the fruit is maintained for a longer period as well.

3. Production of medicinal plants:

(a) Golden rice: in this rice, beta carotene content has been increased. There are three genes responsible for beta carotene. All three genes were transferred into the plant to increase beta carotene content. Once golden rice is consumed by humans – beta carotene is converted into vitamin A in the liver. Since beta carotene is yellow in color – this resulted in yellow color of the rice grain. Use of golden rice prevents NIGHT BLINDNESS.

4. Plants with herbicide resistance: weeds are considered as unwanted plants that compete with the main crop for solar energy and nutrients from the soil. Weeds can be removed manually by uprooting, but it is a very time consuming and laborious process. What if we make plants resistant to herbicides? It means, if we use a herbicide, it would kill only the weeds and not the main crop / plant.

5. Production of ornamental plants – rose, tulips, sunflower, etc.

Genetic engineering is used for the improvement in colors. Colors are controlled by anthocyanin. Alternation in anthocyanin production leads to the improvement in colors. This way, we can increase in the aesthetic value of the plant.

Biotechnology applications: Advantages of GM crops:

• Pest resistance
• Herbicide resistance
• Soil fertility – GM crops use soil nutrient resources in a more judicious manner.
• Post harvest losses: GM crops help decrease the post harvest loss by increasing the shelf life.
• Medicinal value: include medicinal properties 
• Phytoremediation: GM plants can be used to control metal pollution. These plants can accumulate toxic metals from the soil or from the environment.
• Alternatives for industry: GM crops can be grown for the formation of many pharmaceutical products / medicines.

Disadvantages of GM crops:

Nature:

• Harm to non-target organisms: No natural evidence is present till now, but if considering a possibility, like Bt plants and milk weed vines and monarch butterfly. This butterfly is beneficial for nature because it is involved in pollination. Suppose, if the pollen grains from Bt cotton falls on milk weed vines. The pollen grains can be taken by the larvae of the monarch buttefly. By eating Bt cotton pollen grains, they are getting cry genes, which will result in the killing of the larvae. So, monarch butterfly is a non-target insect which can get killed.
• Transfer of herbicide resistance: GM crop with herbicide resistance may cross with the wild variety. This way the wild variety can get the herbicide resistance gene. So, these weeds would be resistant to herbicides and difficult to remove. Such weeds are called SUPER WEEDS.
• Development of resistant varieties of pest: GM crops are developed against pest resistance. But after some time, the pests can alter themselves and become resistant to these GM crops. For example, DDT was found to be very effective against insect pests, but after certain period of time, many DDT resistant varieties of insect pests developed.

Biotechnology applications in Humans:

• Transfer of antibiotic resistance to intestinal microbes: GM crops are developed which could be antibiotic resistant. If such genes are transferred to the intestinal microbes – these microbes would be difficult to remove with the use of antibiotics.
• Allergy: humans can develop allergic response to the transgenic products.
• Economic concern: GM crops/products are costlier.

TRANSGENIC ANIMALS

Q8. Which of the following steps are not involved in the formation of transgene animals?

1. Selection of desirable gene
2. Isolation of gene and transfer to a suitable vector
3. Transfer of recombinant vector to animal’s germ cells
4. Transfer of recombinant vector to animal’s somatic cells

Ans: 3

If a gene is transferred to the germ cell means all the animal cells will have transgene with it. If it is done with somatic cells then only few cells will be having that transgene. In case of transgenic animals, the vector used is generally VIRUSES.

Biotechnology applications: APPLICATION OF TRANSGENIC ANIMALS

1. Medicinal compounds / recombinant products

For example: alpha-1 antitrypsin is used for the treatment of EMPHYSEMA. The gene responsible for alpha-1 antitrypsin can be transferred to some animal. The animal will then produce that product for us.

CFTR protein: (Cystic Fibrosis Transmembrane Regulator Protein) for the treatment of cystic fibrosis.

2. Vaccine safety: whatever compound is being produced is whether safe or not for the human use. So, vaccine safety testing is done with these transgenic animals. For example, mice are being developed for use in testing the safety of vaccines before they are used on humans. Transgenic mice are used to test the safety of the polio vaccine.

3. Chemical safety testing: it is done to test whether a chemical is having any effect on gene. In this case, the gene is exposed to the chemical. After exposure, the gene is transferred to the lab animal to see its expression. If the gene is having changes, it means the chemical is not safe for the gene. On the other hand, if gene is expressing normally, it means that particular chemical is safe for that gene.

4. Disease models: if a disease is present in a particular animal say, humans and if someone wants to study about that disease – he will develop that disease in lab animals to study. So, these lab animals will be called as disease models.

5. Gene expression: transgenic animals can be used to study the expression of genes. For example, if we want to study the gene expression of a human gene – we can transfer that gene in an animal and see how that gene is expressed and how we can control the gene expression.

6. Spare parts: it means if any organ of the body is damaged, it can be transplanted using certain transgenic animals. For example, pig heart is very close to human heart in terms of its function. Other organ like liver can also be transplanted. In other words, transgenic animals are developed as a source of organ donor.

7. Food value enhancement: using transgenic method, the size of the animals is increased to increase its food value. For example, goat size can be increased, which is consumed by humans.

VARIOUS EXAMPLES OF TRANSGENIC ANIMALS

1. Transgenic fish: for the first time, transgenic SALMON was developed. This was done by modifying the sperm (modified by having an extra copy of GH gene). The modified sperm was used to fertilize the ovum. The transgenic SALMON grows faster and attains larger size than the normal SALMON. Transgenic SALMON was the first animal to be used for food value purpose. Similarly, other transgenic fishes have been developed making them resistant to viruses. In this case, the defective copy of the viral genome was integrated with the genome of the fish.

2. Transgenic birds: In poultry, avian lucosis virus (ALV) infects the birds. These birds have been made resistant against ALV by introducing a defective copy of viral genome to the bird’s genome.

3. Transgenic mice: 95% of the transgenic animals are mice. Why mice has been taken because they have a short gestation period leading to the production of progeny. Besides, the life span is short – it means more generations can be studied. They have been developed as disease models, chemical toxicity symptoms, etc.

4. Transgenic rabbit: they have been developed mostly for MOLECULAR FARMING. In other words, they are acting as bioreactors for obtaining the various biological products. The desired gene is transferred in the rabbit cells and then the product is harvested. These rabbits are being used for the production of products like alpha-1 antitrypsin, TPA (Tissue Plasminogen Activator), etc.

5. Transgenic sheep: developed by fusing the foreign gene with the gene of mammary tissue (beta-lacalbumin gene). Transgenic sheep has been developed to produce alpha-1 antitrypsin, blood clotting factors. Transgenic sheep has also been developed with an extra copy of GH gene to increase the rate of growth and for attaining a size larger than the normal size.

6. Transgenic goat: In this case also the transgene is integrated with the mammary tissue gene. Example: Nexia biotech isolated the gene responsible for spider web protein and integrated this gene with the mammary tissue gene. As a result, the goat milk produced milk having spider web protein. This spider web protein has been used for making a special type of dress material for army personnel. It has been named as BIOSTEEL because of its strength.

7. Transgenic cow: In 1997, ROSIE, a transgenic cow was developed. It was having the alpha-lactalbumin gene, which resulted in the production of alpha-lactalbumin protein. And this protein was present in milk at 2.4 gram/liter. Because of the presence of this protein, the milk has become more nutritious.

8. Transgenic pig: it has been developed for the food value and also for the suitable organ-spare source.

9. Transgenic dog: developed with better olfactory power. Such dogs are named as DOGIE.

10. Transgenic monkey: it was developed in the US by the Oregon group. They isolated gene from JELLY FISH responsible for green fluorescent protein (GFP). This gene was introduced in the oocyte of the monkey tbihrough MICROINJECTION. After the GFP gene was transferred successfully in the oocyte then the sperms were introduced using ICSI (intra cytoplasmic sperm injection) for fertilization. The transgenic monkey was named as ANDi (invert DNA). This experiment suggested that these primates can also be used as disease models for diseases like PKU, Alzheimer, etc.

BIOTECHNOLOGY IN THE FIELD OF MEDICINE

• Used in producing the therapeutic drugs
• These drugs are safe to use
• These drugs are immunologically safe
• About 30 drugs have been approved for the human use. In India, 12 drugs are being used.

INSULIN

• Insulin is required for the treatment of diabetes mellitus (IDDM)
• In the beginning, insulin was isolated from the pancreatic cells of dog.
• Later on, bovine insulin obtained from cow and buffalo was used then porcine insulin obtained from pig was used.
•  Obtaining insulin from animal sources was very costly because the process of extraction was tedious and only a small amount of insulin would be obtained. Besides, the body’s immune system would response against these insulin sources. In other words, they were not immunologically safe

Insulin when it is produced first – it is in the form of PROINSULIN, which is inactive in its form. In proinsulin, there are three polypeptide chains: A, B, and C

Polypeptide chain A – made up from 21 amino acids

Polypeptide chain B – made up from 30 amino acids

Polypeptide chain C – made up from 30 amino acids

The active form of insulin is made only when the chain C is removed from the proinsulin. This problem was solved by an American company, Eli Lily. The company manufactured polypeptide A and polypeptide B separately. This was done by separating the polypeptide A and polypeptide B gene responsible for insulin formation. The scientist transferred these genes with galactosidase gene in E.coli. Both polypeptide A and B were linked together. This insulin has been named as HUMULIN – it acts in a similar way as insulin.

hGH: Human Growth Hormone: When hGH gene was made cDNA by reverse transcriptase and then introduced into the bacteria – it failed to express itself. When it was non functional then it was treated with specific restrictoion endonuclease. This RE separated the part of cDNA responsible for coding 24 amino acids. The remaining cDNA was linked with a small obligonucleotide (synthesized synthetically), which would code for the same 24 amino acids. So, the cDNA contained two parts: first part as synthetic DNA and the second part as original cDNA. When this DNA was transferred in bacteria – this expressed successfully.

 

Gene Therapy:

There are hereditary diseases, which are caused due to defective genes. If we could rectify such genes after birth then that would come under gene therapy. In therapy, we can have various options:

(a) We can introduce a normal gene in the body cell. So, the cells would have both normal as well as defective genes. In some cases, this works successfully, but in several other cases, it does not work. In that case, we need to remove the defective gene and introduce a normal gene at the same position. Now, at what level gene therapy is being performed? It can be carried out at the level of germ cells – called germ cell gene therapy. And if gene therapy is performed at the somatic cell level then it will be called as somatic cell gene therapy. In case of animals, we can use both germ cell and somatic cell gene therapy. But when we talk about humans, only somatic cell gene therapy is allowed. Germ cell gene therapy is not allowed in humans due to various ethical issues. Under gene therapy, we will discuss the disease, SCID (Severe Combined Immune Deficiency).

SCID is caused due to the absence of ADA enzyme (Adenosine deaminase enzyme). Due to defective ADA gene, ADA enzyme is not produced.

Line of treatments:

• Bone marrow transplantation – to produce normal lymphocytes.
• Enzyme therapy – meaning giving the enzyme from outside.
• Both the methods mentioned are costly and effectiveness is not sure.

Gene therapy for SCID: in 1990, a 4 year old girl was suffering from SCID. Scientists separated lymphocytes from the bone marrow. In the lymphocyte cells, ADA cDNA was introduced using the retrovirus and helper virus so that the ADA cDNA could get integrated with the ADA cell genome. In other words, these lymphocyte cells have been transformed. The transformed lymphocytes were cultured because the life cycle of lymphocyte is short and therefore periodic injection of lymphocytes needs to be done.

MOLECULAR DIAGNOSIS

Under this category – RDT, PCR, and ELISA techniques are used to diagnose a disease at an early stage.

BIOPATENT – patent is related to copyright.

Biopatent can be awarded for any gene sequence, altered gene sequence, transgenic organism, any new techniques, any alteration in the technique. Patent can also be given for any traditional knowledge.

BASMATI—in India, 27 documented varieties of rice are being grown in India. One American company got a new variety of basmati patented. The company crossed basmati variety with a dwarf variety. The progeny formed were named BASMATI and patented. However, this patent was discarded.

In India, we have Indian Patent Bill -2 has been passed by the parliament to tackle any issue related to the biopatent. Besides, all work under genetic engineering is taken care by the GEAC (Genetic Engineering Approval Committee).

BIOPIRACY: Use of patented biological products without giving the royalty to the person / company holding the patent right. In other words, it is illegal use of biopatent products.

BRAZZEIN: it is obtained from the plant PENTADIPLANDRA BRAZZIENA. A number of African countries have been using it. One American company isolated the gene responsible for the formation of brazzein and transferred it to the maize plant. The seeds of maize contain brazzein – the company got it patented.

Bioterror /Biowar – to create terror by using biological agents or organisms. Anthrax spores

Biowars – -use of bio weapons. Production of disease causing spores; cost is very low; can be released silently; destruction is very high.

BIOETHICS: ethics means certain norms set by the society to be followed. If the same is applied with a living organism, it will come under bioethics.

Bioethics is important because genetic engineering dilutes the concept of species. Genetic engineering is cruelty against animals. They are being used as factories.