Southern blotting:-
- Discovered by Edward M. Southern at Edinburgh University in 1970.
- It is designed to locate a particular sequence of DNA within a complex mixture. For example, it could be used to locate a particular gene within an entire genome.
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Western Blotting:-
> Western blot is performed by using polypropylene gel electrophoresis. SDS-PAGE allows protein samples to be separated and transferred to a solid support, such as nitrocellulose (NC) or polyvinylidene difluoride (PVDF) membrane.
> The solid support can absorb the protein and keep its biological activity unchanged. The transferred solid support membrane is called a blot and is treated with a protein solution to block the hydrophobic binding site on the membrane.
> The membrane is treated with the antibody (primary antibody) of the target proteins.
> Only the proteins to be studied can specifically bind to the primary antibody to form an antigen-antibody complex.
> After the primary antibody is washed and removed, only the position of the target protein binds to the primary antibody.
> The primary antibody-treated membranes are treated with a labeled secondary antibody after washing.
> After treatment, the labeled secondary antibody that binds to the primary antibody forms an antibody complex that can indicate the location of the primary antibody, both the location of the protein being studied.
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Antisense - RNA:- It is single-stranded RNA molecule that can bind and inhibit specific mRNA translation to protein.
Principle:-
Sense RNA:-
> messenger RNA (mRNA) is single-stranded.
> Its sequence of nucleotides is called "sense" because it results in a gene product (protein).
> Normally, its unpaired nucleotides are "read" by transfer RNA anticodons as the ribosome proceeds to translate the message.
Antisense RNA:-
> RNA can form duplexes just as DNA does.
> All that is needed is a second strand of RNA whose sequence of bases is complementary to the first strand.
> The second strand is called the antisense strand because its sequence of nucleotides is the complement of message sense.
Example:-
5´ C A U G 3´ mRNA
3´ G U A C 5´ Antisense RNA
Molecular farming:- It describes the production of recombinant proteins and other secondary metabolites in plants. This technology depends on a genetic transformation of plants.
Cloned Genes in Medicine:-
1. Production of Human Insulin:-
> The human insulin gene is cloned into bacteria (such as Escherichia coli) or yeast.
> These microorganisms produce recombinant human insulin in large quantities.
> It is used to treat diabetes mellitus.
> Recombinant insulin is safer and purer than animal-derived insulin and reduces the risk of allergic reactions.
2. Production of Human Growth Hormone (hGH):-
> The human growth hormone (hGH) gene is cloned into bacteria.
> The bacteria produce large amounts of recombinant hGH.
> It is used to treat growth hormone deficiency (pituitary dwarfism) in children and certain growth disorders in adults.
> Recombinant hGH avoids the risk of disease transmission associated with hormone extracted from human pituitary glands.
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Uses of Cloned Genes in Industries:- Cloned genes are widely used in biotechnology industries to produce valuable proteins and enzymes on a large scale.
1. Production of Human Insulin:-
> The human insulin gene is cloned into bacteria such as Escherichia coli or yeast.
> These microorganisms produce large quantities of recombinant human insulin.
> The insulin is purified and used for the treatment of diabetes mellitus.
> Industrial significance: Safe, highly pure, and cost-effective production compared to insulin extracted from animal pancreas.
2. Production of Chymosin (Rennin) for Cheese Industry:-
> The chymosin gene (from calf stomach) is cloned into microorganisms such as bacteria, yeast, or fungi.
> These genetically modified microorganisms produce recombinant chymosin, an enzyme used for milk coagulation in cheese making.
> Industrial significance: Produces high-quality cheese, reduces dependence on animal sources, and is suitable for large-scale commercial production.
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Herbicide-Resistant Plants:- Herbicide-resistant plants are genetically engineered or conventionally bred plants that can survive the application of a specific herbicide, allowing farmers to control weeds without damaging the crop.
Definition:- A herbicide-resistant plant is a crop that possesses genes enabling it to tolerate one or more herbicides that would normally kill or inhibit plant growth.
How Herbicide Resistance is Achieved:-
> Genetic engineering (Transgenic approach):
> Introduction of a resistance gene from another organism.
> Example: Transfer of the CP4 EPSPS gene from Agrobacterium to provide glyphosate resistance.
> Mutation breeding or conventional breeding:
> Selection of naturally occurring or induced herbicide-resistant mutants.
> The resistance trait is incorporated into commercial crop varieties through breeding.
Advantages:-
> Effective weed control.
> Higher crop yield due to reduced weed competition.
> Reduced labor and cultivation costs.
> Facilitates conservation tillage and no-till farming, reducing soil erosion.
> Simplifies weed management.
Limitations:-
> Development of herbicide-resistant weeds with repeated herbicide use.
> Increased dependence on a single herbicide.
> Potential environmental concerns due to overuse of herbicides.
> Higher cost of genetically modified seeds.
> Possible gene flow to related wild species.
Examples:-
Roundup Ready Soybean:- Resistant to glyphosate due to the CP4 EPSPS gene from Agrobacterium.
LibertyLink Cotton:- Resistant to glufosinate due to the bar gene from Streptomyces hygroscopicus.
Clearfield Rice:- Resistant to imidazolinone herbicides through mutation in the ALS (acetolactate synthase) gene (developed by mutation breeding, not transgenic).
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Transcription in prokaryotes:-
Introduction:-
• It is the process of copying genetic information from one strand of the DNA into RNA.
• Here, adenine pairs with uracil instead of thymine.
• Both strands are not copied during transcription, because:-
- The code for proteins is different in both strands. This complicates the translation.
- If 2 RNA molecules are produced simultaneously this would be complimentary to each other, hence form a double stranded RNA. This prevents translation.
Transcription Unit:-
• It is the segment of DNA between the sites of initiation and termination of transcription. It consists of 3 regions:-
- A promoter (Transcription start site):- Binding site for RNA polymerase.
- Structural gene:- The region between promoter and terminator where transcription takes place.
- A terminator:- The site where transcription stops.
• The DNA- dependent RNA polymerase catalyzes the polymerization only in 5’→3’direction.
• 3’→5’ acts as template strand and 5’→3’ acts as coding strand.
3’-ATGCATGCATGCATGCATGCATGC-5’ template strand.
5’-TACGTACGTACGTACGTACGTACG-3’ coding strand.
Transcription unit and gene:-
• Gene:- Functional unit of inheritance. It is the DNA sequence coding for RNA molecule.
• Cistron:- A segment of DNA coding for a polypeptide.
• Structural gene in a transcription unit is of 2 types:-
i. Monocistronic structural genes (split genes):- It is seen in eukaryotes. Here, the coding sequences exons are interrupted by introns.
ii. Polycistronic structural genes:- It is seen in prokaryotes. Here, there are no split genes.
Reverse Transcription:- It is the process in cells by which an enzyme makes a copy of DNA from RNA. The enzyme that makes the DNA copy is called reverse transcriptase and is found in retroviruses, such as the human immunodeficiency virus (HIV).
Steps of transcription in prokaryotes:-
i. Initiation:- Here, the enzyme RNA polymerase binds at the promoter site of DNA. This causes the local unwinding of the DNA double helix. An initiation factor (σ factor) present in RNA polymerase initiates the RNA synthesis.
ii. Elongation:- The RNA chain is synthesized in the 5’-3’ direction. In this process, activated ribonucleoside triphosphates (ATP, GTP, UTP & CTP) are added. This is complementary to the base sequence in the DNA template.
iii. Termination:- A termination factor (ρ factor) binds to the RNA polymerase and terminates the transcription.
NOTE:- In bacteria (Prokaryotes) transcription and translation can be coupled (Translation can begin before mRNA is fully transcribed) because:-
- mRNA requires no processing to become active.
- Transcription and translation take place in the same compartment (no separation of cytosol and nucleus).
Polysome or Polyribosome or Ergosome:- It is a group of ribosomes bound to an mRNA molecule like “beads” on a “thread”. It consists of a complex of an mRNA molecule and two or more ribosomes that act to translate mRNA instructions into polypeptides.
Transcription in eukaryotes:-
In eukaryotes, there are 2 additional complexities-
i. There are 3 RNA polymerases:-
• RNA polymerase I:- Transcribes rRNAs (28S, 18S & 5.8S).
• RNA polymerase II:- Transcribes mRNA.
• RNA polymerase III:- Transcribes tRNA, 5S rRNA and snRNAs.
ii. The primary transcripts (hnRNA):- It contain both the exons and introns and is non-functional. Hence introns have to be removed. For this, it undergoes splicing process.
Heterogenous RNA Processing:- The hnRNA is the collective term for the unprocessed mRNA (pre-mRNA) molecules in the nucleus. It contain both the exons and introns and is non-functional. Hence introns have to be removed. For this, it undergoes the following processes-
i. Splicing:- From hnRNA introns are removed by the spliceosome and exons are joined together.
ii. Capping:- Here, a nucleotide methyl guanosine triphosphate (cap) is added to the 5’ end of hnRNA.
iii. Tailing (Polyadenylation):- Here, adenylate residues (200-300) are added at 3’-end. It is the fully processed hnRNA, now called mRNA.
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Nif genes:-
1. Introduction:-
> Nif means:-
Ni = Nitrogen
f = fixation
> These are genes encoding enzymes involved in the fixation of atmospheric nitrogen.
> Nif genes also encode a number of regulatory proteins involved in nitrogen fixation.
> The nif genes are found in both free-living and symbiotic nitrogen-fixing bacteria.
> The primary enzyme encoded by the nif genes is the nitrogenase.
2. Regulation of Nif genes:- In most bacteria, regulation is done by NifA protein.
i. When there is not enough fixed nitrogen, NtrC triggers NifA expression, and NifA activates the nif genes.
ii. When there is not enough fixed nitrogen, NifL inhibit NifA expression, and nif genes remain inactive.
Reversible ADP-ribosylation:- It is an additional regulation mechanism found in Rhodospirillum rubrum. Reversible ADP-ribosylation of a specific arginine residue in the nitrogenase complex. When reduced nitrogen is present, DraG and DraT catalyze the ribosylation of arginine residue in the nitrogenase. It causes a barrier in the electron transfer flow and thereby inactivates nitrogenase activity.
3. Expression of Nif genes:-
> There are total 20 nif genes.
> nifH, nifD, and nifK:- They encode the nitrogenase subunits.
> nifE, nifN, nifU, nifS, nifV, nifW, nifX, nifB, and nifQ:- They encode proteins involved the assembly and incorporation of Fe and Mo atoms into the nitrogenase subunits.
> nifF and nifJ:- They encode proteins related to electron transfer taking place in the reduction process.
> nifA and nifL:- They are regulatory proteins in charge of regulating the expression of the other nif genes.
Nitrogen fixation:- It is a chemical process by which molecular nitrogen found in the air is converted into ammonia or related nitrogenous compounds.
1. Types of nitrogen fixation:-
a. Physical Nitrogen Fixation
b. Biological Nitrogen Fixation
a. Physical Nitrogen Fixation:-
i. Natural Nitrogen Fixation:- Under the influence of lightning and thunder, N2 and O2 of the air react to form nitric oxide (NO). The nitric oxides are again oxidized with oxygen to form nitrogen peroxide (NO2).
ii. Industrial Nitrogen Fixation:- Ammonia is produced industrially by direct combination of nitrogen with hydrogen (obtained from water) at high temperature and pressure. Later, it is converted into various kinds of fertilizers, such as urea etc.
b. Biological Nitrogen Fixation:- The conversion of atmospheric nitrogen into the nitrogenous compounds by living organisms is called biological nitrogen fixation. Only prokaryotes can fix nitrogen. Nitrogen fixation require anaerobic conditions because oxygen inactivates nitrogenase enzyme.
Hence for obligate anaerobes nitrogen fixation is easy, but in case of facultative anaerobes the nitrogen fixation occurs only in anaerobic conditions. In case of obligate aerobes the oxygen level inside the cell must be kept low for nitrogen fixation.
2. Nitrogen Fixers (Diazotrophs):- Among the earth’s organisms, only some prokaryotes like bacteria and cyanobacteria can fix atmosphere nitrogen. They are called nitrogen fixers or diazotrophs. They fix about 95% of the total global nitrogen fixed annually by natural process.
a. Asymbionts (Free living)
b. Symbionts
a. Asymbionts (Free living):-
i. Bacteria:- They add up to 10-25 kg, of nitrogen/ha/annum.
> Azotobacter (Aerobic, Saprophytic)
> Beijerinckia (Aerobic, Saprophytic)
> Clostridium (Anaerobic, Saprophytic)
> Desulphovibrio (Chemotrophic)
> Rhodopseudomonas (Photoautotrophic)
> Rhodospirillum (Photoautotrophic)
> Chromatium (Photoautotrophic)
ii. Blue Green Algae (Cyanobacteria):- Heterocysts are the special cells that fix nitrogen. They add 20-30 kg Nitrogen/ha/annum.
> Nostoc
> Anabaena
> Aulosira:- A. fertilissima is the most active nitrogen fixer in Rice fields.
> Cylindrospermum:- It is active in sugarcane and maize fields.
> Trichodesmium
b. Symbionts:- Live in close symbiotic association with other plants.
i. Blue Green Algae (Cyanobacteria):-
> Nostoc and Anabaena:- They are common symbionts in lichens, Anthoceros, Azolla and cycad roots.
> Anabaena azollae:- It is found in fronds of Azolla pinnata (a water fern). It is often inoculated to Rice fields for nitrogen fixation.
ii. Bacteria:-
> Rhizobium:- It is aerobic, gram negative nitrogen fixing bacterial symbionts of legume roots. Sesbania rostrata has Rhizobium in root nodules and Aerorhizobium in stem nodules.
> Frankia:- It is symbiont in root nodules of many non-leguminous plants like Casuarina and Alnus.
> Xanthomonas and Mycobacterium:- They occur as symbiont in the leaves of some members of the families Rubiaceae and Myrsinaceae (e.g., Ardisia).
3. Rhizobium Nitrogen Fixation:-
> Rhizobium bacteria:-
i. Free living
ii. Gram negative
iii. Aerobic
iv. Soil bacteria
> Rhizobium becomes anaerobic upon entry into roots.
> Leghaemoglobin (legHb or symbiotic Hb):-
- It is a pink coloured pigment.
- It occurs in the root nodules of leguminous plants.
- It acts as an oxygen scavenger. It provides anaerobic conditions for the nitrogenase enzyme and protects the enzyme from inactivation.
> Two main steps:-
a. Nodule formation
b. Nitrogen fixation
a. Nodule formation:- Root nodule formation is initiated, when the soil contains a low level of nitrogen. Steps of nodulation are:
i. Aggregation:- Roots of legumes secrete flavonoids, which attracts rhizobia towards the root. Rhizobia aggregate around root hairs.
ii. Developmental changes:- Rhizobia secrete nod factors, which causes stimulate many developmental changes:
- Membrane depolarization
- Curling of root hairs
- Cell division in the root cortex
- Intracellular calcium movement
iii. Infection thread:- The nod factor attaches to receptors present on the plasma membrane of the root hairs, which leads to the formation of the infection thread.
iv. Entry:- Infection thread provides the passage to bacteria to enter epidermal cells. Rhizobia then enter cortex cells, each bacterium gets surrounded by a plant-derived membrane known as symbiosome.
v. Nodulation:- Nodule formation is initiated by chemicals produced by rhizobia. It is a result of calcium dependent signal transduction pathway, which triggers biochemical changes leading to cell division and nodule formation. Cytokinin also plays an important role in nodules formation.
vi. Bacteroids:- Within nodules, bacteria get differentiated into bacteroids, which fix nitrogen. The Rhizobia stop dividing, loose cell wall and become nitrogen fixing cells as led bacteroids . Vascular tissues are developed for nodules for exchange of nutrients.
b. Nitrogen fixation:-
- The nodule serves as site for N2 fixation.
- Nodule contains nitrogenase and leghaemoglobin.
- The nitrogenase has 2 components:
i. Molybdoferredoxin (Mo-Fe protein)
ii. Azoferredoxin (Fe-protein).
- The free di-nitrogen first bound to MoFe protein and is not released until completely reduced to ammonia.
- In this process ferredoxin serves as an electron donor to Fe-protein (nitrogenase reductase) which in turn hydrolyzes ATP and reduce MoFe protein, the MoFe protein in Turn reduce the substrate N2. The electrons and ATP are provided by photosynthesis and respiration of the host cells.
- Many intermediates are formed to form ammonia (NH3).
Dinitrogen → Hydrazine → Diamine → Ammonia
- Ammonia (NH3) is immediately protonated at physiological pH to form ammonium ion (NH4+). As NH4+ is toxic to plants, it is rapidly used near the site of generation to synthesize amino acids.
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Male Sterility:- When the pollens of a plant are dead and inactive, while the female gametes are alive and active, it is called male sterility. This occurs very rarely. This occurs due to mutation. There are three types of male sterility: -
1. Genetic Male Sterility (GMS)
2. Cytoplasmic Male Sterility (CMS)
3. Cytoplasmic Genetic Male Sterility (CGMS)
1. Genetic Male Sterility (GMS):- When male sterility is controlled by a recessive gene present in the nucleus, it is called genetic male sterility (GMS).
MS gene = For male fertility
ms gene = For male sterility
MS MS = Male fertile plant
MS ms = Male fertile plant
ms ms = Male sterile plant
Maintenance:- For maintaining GMS, male sterile plants are cross heterozygous male fertile plants. As a result, 50 percent male sterile plants are obtained in each generation.
Application in hybrid seed production:- For hybrid seed production, the male sterile line is used as the female parent and crosses with the appropriate male parent line. Hybrid seeds are produced using GMS in Castor and Pigeon pea crops.
EGMS (Environment sensitive GMS):- The environment has a great impact on genetic male sterility. Genetic male sterility is determined by photo period and temperature. This property is used in hybrid seed production in paddy crop. Thus, there are two types of EGMS:
i. TGMS (Temperature sensitive GMS)
ii. PGMS (Photoperiod sensitive GMS)
i. TGMS (Temperature sensitive GMS):- Temperature has an effect on genetic male sterility in paddy crop. At temperatures below 28 ° C, paddy plants are completely male fertile while at temperatures above 30 °C, paddy plants are completely male sterile.
Maintenance of TGMS line:- For this, paddy plants are grown in an area where the day temperature remain below 28 ° C. At this temperature, paddy plants are fully male fertile. Now allow self-pollination in plants, resulting in the production of TGMS line seeds and then plants.
Hybrid Seed Production:- For this, paddy plants are grown in an area where the temperature is above 30 ° C. At this temperature paddy plants are fully male sterile which can be used as female parent. Now these male sterile female parent plants are crossed with the appropriate male parent plant, resulting in the production of hybrid seeds.
ii. PGMS (Photoperiod sensitive GMS):- Photo period also has an effect on genetic male sterility in paddy crop. Paddy plants are fully male fertile at 10 hours of light period, while at 14 hours of light, paddy plants are completely male sterile.
Maintenance of PGMS line:- For this, paddy plants are grown in an area where light period remain available for 10 hours. Paddy plants are fully male fertile in this condition. Now allow self-pollination in plants, resulting in the production of PGMS line seeds and then plants.
Hybrid Seed Production:- For this, paddy plants are grown in an area where the light period lasts for 14 hours. Paddy plants are fully male sterile in this condition which can be used as female parent. Now these male sterile female parent plants are crossed with the appropriate male parent plant, resulting in the production of hybrid seeds.
2. Cytoplasmic Male Sterility (CMS):- When male sterility is controlled by cytoplasm, it is called cytoplasmic male sterility (CMS). When fertilization occurs, only the nucleus comes from the male parent, while both the nucleus and cytoplasm come from the female parent. So when the male sterile female parent is crossed with the male parent, all the plants produced from the hybrid seeds are male sterile as shown in the diagram below.
Hybrid Seed Production:- CMS cannot be used for hybrid seed production in crops in which seed is of economic importance. But in crops where a somatic part is of economic importance rather than seed, CMS is used for hybrid seed production. Such as onion, garlic, carrot, radish etc.
3. Cytoplasmic Genetic Male Sterility (CGMS):- When male sterility is controlled by both nucleus and cytoplasm, it is called cytoplasmic genetic male sterility (CGMS). Cytoplasm is the main controller. The dominant gene present in the nucleus is called restorative gene, which acts to restore male fertility by preventing the male sterile effect of cytoplasm. We denote this as R letter. While r is the inactive gene.
R gene = For male fertility
r gene = For male sterility
RR = Male fertile plant
Rr = Male fertile plant
rr = Determined by cytoplasm.
Different combinations of nucleus and cytoplasm are given in the following diagram: -
Hybrid Seed Production:- CGMS is most commonly used in hybrid seed production. A three line system has been developed for hybrid seed production using CGMS which consists of the following three lines:
i. A – line:- It is a male sterile line that is used as a female parent.
ii. B – line:- It is a male fertile line which is used as a male parent. It is the maintainer line that works to maintain the male sterile A - line.
iii. R – line:- It is a male fertile line which is used as a male parent. It is a restore line that works to restore the male fertility in hybrid plants.
To maintain the A-line, its cross is made with the B-line. For hybrid seed production, the A-line crosses with the R-line as shown in the diagram below.
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Antisense - RNA:- It is single-stranded RNA molecule that can bind and inhibit specific mRNA translation to protein.
Principle:-
Sense RNA:-
> messenger RNA (mRNA) is single-stranded.
> Its sequence of nucleotides is called "sense" because it results in a gene product (protein).
> Normally, its unpaired nucleotides are "read" by transfer RNA anticodons as the ribosome proceeds to translate the message.
Antisense RNA:-
> RNA can form duplexes just as DNA does.
> All that is needed is a second strand of RNA whose sequence of bases is complementary to the first strand.
> The second strand is called the antisense strand because its sequence of nucleotides is the complement of message sense.
Example:-
5´ C A U G 3´ mRNA
3´ G U A C 5´ Antisense RNA
Antisense technology:-
> When mRNA forms a duplex with a complementary antisense RNA sequence, translation is blocked.
> This may occur because the ribosome cannot gain access to the nucleotides in the mRNA or because the duplex RNA is quickly degraded by ribonucleases in the cell.
> With recombinant DNA methods, synthetic genes (DNA) encoding antisense RNA molecules can be introduced into the organism.
Applications of Antisense technology:-
1. Treatment of viral infection:-
> Antisense oligonucleotides are selective and highly specific and binds with targeted viral mRNA and down regulate the expression of viral proteins, thus inhibit the virus replication.
> Thus this antisense technology can be used to design therapeutic compounds targeted to specific mRNA to treat various viral diseases.
> Vitravene (fomivirsen):- It is the first antiviral Antisense drugs used to treat cytomegalovirus retinitis. Other potential targets of antisense drugs include human papillomavirus, HIV, hepatitis-B virus, influenza A virus, and herpes simplex virus (HSV).
2. Treatment of cancer:-
> As antisense drugs are less toxic than conventional drugs, many antisense oligonucleotides are currently investigated to treat various cancers in humans or for the deactivation of oncogenes.
> For the first time an antisense oligonucleotide in combination with cisplatin was approved to treat bladder cancer.
3. Gene therapy:-
> Antisense oligonucleotides are being used in genetic research to treat various genetic disorders.
> In genetic disorders, some point mutations resulting in the formation of a defective mRNA and then defective protein.
> Antisense oligonucleotides blocks the formation of defective mRNA and then defective proteins.
> β-thalessemia:- A genetic blood disorder can be completely treated using antisense technology in which O-alkyl oligonucleotides or morpholino-oligonucleotides have been used.
4. Medical applications:-
> Antisense technology is used to investigate protein function in the living brain to study central nervous system (CNS) proteins such as transmembrane receptors, ion channels, transporters, G proteins, and growth factors.
> Antisense oligonucleotides can be used to inhibit expression of a particular enzyme. For eg. Inhibition of acetyl cholinesterase enzyme is the molecular target for the treatment of diseases like Alzheimer’s disease.
> In inflammatory diseases, antisense oligonucleotides is used to demonstrate the relative importance of various signaling components at the molecular level.
> A novel respirable antisense drug called RASONS is used to treat various respiratory diseases including asthma, influenza, bronchitis, pulmonary fibrosis, pneumonia, and lung cancer.
> Antisense oligonucleotide can be used target specific membrane component that influences the pathophysiological mechanisms in renal and cardiovascular disorders.
5. Flavr Savr tomatoes:-
> Most tomatoes that have to be shipped to market are harvested before they are ripe. Otherwise, ethylene synthesized by the tomato causes them to ripen and spoil before they reach the customer.
> Transgenic tomatoes have been constructed that carry in their genome an artificial gene (DNA) that is transcribed into an antisense RNA complementary to the mRNA for an enzyme involved in ethylene production. These tomatoes make only 10% of the normal amount of the enzyme.
> The goal of this work was to provide supermarket tomatoes with something closer to the appearance and taste of tomatoes harvested when ripe.
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Socio-Economic Aspects of Biotechnology:- Biotechnology is the application of living organisms, cells, or biological systems to develop useful products and technologies for agriculture, medicine, industry, and environmental management. It has significantly influenced socio-economic development by improving food security, healthcare, employment, industrial productivity, and environmental sustainability. However, it also raises ethical, legal, and social concerns.
Socio-Economic Aspects of Biotechnology:-
1. Improvement in Agriculture:-
> Development of high-yielding and disease-resistant crop varieties.
> Production of drought-, salinity-, and pest-resistant crops through genetic engineering.
> Reduced dependence on chemical pesticides and fertilizers.
> Improved nutritional quality of crops (e.g., Golden Rice enriched with Vitamin A).
> Increased farmers' income through higher productivity.
2. Healthcare Benefits:-
> Production of vaccines, antibiotics, insulin, growth hormones, and monoclonal antibodies.
> Early diagnosis of diseases using molecular diagnostic tools such as PCR and ELISA.
> Gene therapy and stem cell therapy for treating genetic disorders.
> Lower mortality rates and improved quality of life.
> Rapid development of vaccines during disease outbreaks.
3. Industrial Development:-
> Production of enzymes, biofuels, organic acids, and biodegradable plastics.
> Improved efficiency in food, textile, paper, leather, and pharmaceutical industries.
> Reduced production costs through microbial fermentation.
> Promotion of environmentally friendly industrial processes.
4. Employment Generation:-
> Creation of jobs in biotechnology research laboratories, pharmaceutical industries, agriculture, food processing, and environmental management.
> Increased demand for skilled professionals in molecular biology, bioinformatics, genetic engineering, and biotechnology.
> Growth of biotechnology startups and entrepreneurship opportunities.
5. Economic Growth:-
> Biotechnology contributes significantly to national GDP through agricultural exports, pharmaceuticals, and industrial biotechnology.
> Increased foreign investment in biotechnology research and development.
> Promotion of innovation and commercialization of new technologies.
> Strengthening of the bioeconomy.
6. Environmental Sustainability:-
> Bioremediation helps clean polluted soil and water.
> Biofertilizers and biopesticides reduce environmental pollution.
> Development of renewable biofuels decreases dependence on fossil fuels.
> Conservation of biodiversity through tissue culture and cryopreservation.
7. Food Security:-
> Increased food production to meet the needs of a growing population.
> Enhanced shelf life and nutritional quality of food products.
> Reduction in post-harvest losses through biotechnology-based preservation methods.
8. Ethical and Social Issues:-
> Concerns about the safety of genetically modified (GM) crops.
> Potential health and environmental risks.
> Ethical issues related to cloning, gene editing, and stem cell research.
> Public acceptance varies due to cultural and religious beliefs.
9. Intellectual Property Rights (IPR):-
> Biotechnology innovations are protected by patents.
> Patents encourage research and investment.
> High cost of patented technologies may limit access for poor farmers and developing countries.
> Need for balancing innovation with public welfare.
10. Challenges:-
> High research and development costs.
> Unequal access to biotechnology between developed and developing countries.
> Biosafety and biosecurity concerns.
> Need for proper regulations, public awareness, and skilled manpower.
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Transgenic Plant:-
• Definition:- Stable transformation is achieved when a desired gene is integrated into the genome of a plant, this plant is called transgenic plant.
• These transgenic plants are developed for the following purposes:-
1. Insect Resistance
2. Virus Resistance
3. Seed Protein Quality
4. Gene Silencing
5. Male Sterility
6. Biochemical Production
1. Insect Resistance:-
cry gene transfer:- The cry gene is found in the plasmid of Bacillus thuringiensis (Bt) bacteria. This cry gene makes Crystal protein which is insecticidal.
Ø The cry gene was isolated from the plasmid of Bt and integrated into the cotton genome to form a transgenic plant called Bt-cotton.
Ø This Bt - cotton is resistant to boll worm.
Ø Crystal protein perforates the larva's alimentary canal leading to its death.
2. Virus Resistance:-
cp – gene transfer:- The coat protein is transferred from the TMV virus to the tobacco plant.
Ø The RNA of TMV virus is not come out in the presence of cp-protein. Due to which the tobacco plant becomes resistant to mosaic disease.
Ø Other examples: - Tomato, Alfalfa, Beetroot, Potato
3. Seed Protein Quality:-
SFA8 gene transfer:- In sunflower seeds, the SFA8 gene produces a protein that contains an excess of sulfur-rich amino acids methionine and cysteine. While the proteins present in pea seeds lack methionine and cysteine amino acids.
Ø Therefore the SFA8 gene is transferred to pea seeds.
4. Gene Silencing:-
Slow ripening tomatoes:-
Ø PG (Poly Galacturonase) enzyme digest the pectin. Due to which fruits become soft and fluffy quickly and ripen quickly.
Ø Anti-sense genes are integrated into the genome of tomato plants against genes that code PG enzymes. This inhibits the expression of PG gene and the tomato fruit ripens late.
Ø Antisense gene made against PG gene inhibits its expression, which causes tomato to ripen late and can be easily exported.
5. Male Sterility:-
Producing male sterility:-
Ø Flavonoid are essential for maturation of pollens. If we stop its formation then male sterility can be created in the plant.
Ø The CHS (Chalcone Synthase) enzyme is required for flavonoid synthesis.
Ø Anti-sense genes are integrated into the plant's genome against genes that code CHS.
Ø Absence of a flavonoid causes pollen to become inactive. The plant becomes male sterile.
6. Biochemical Production:-
• PHB (Poly Hydroxy Butyrate):- It is prepared from Acetyl CoA. From this bio-degradable plastic is made.
• The transgenic plant is developed by isolating the phb - B and phb - C genes from the Alcaligenes eutrophus bacterium and transfer to Arabidopsis thaliana. Now PHB is obtained from this plant.
Golden Rice:-
• The genes that form β - carotene are integrated into the embryo of rice within the genome to prepare golden rice.
• 2 genes are isolated from Daffodils plant and 1 gene from Erwinia uredovora bacterium.
• β - carotene is yellow in color, which makes rice yellow.
• β - carotene is the precurssor of vitamin A.
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PCR: Its principles and uses:-
• PCR:- The process of multiplication of DNA segments using DNA polymerase and DNA primers is called PCR.
• Discovery:- The PCR technique was discovered by Kary Mullis in 1985.
• Thermocyclers are used to achieve different temperatures.
• PCR has three main steps -
i. Denaturation:- When dsDNA is heated, both its chains separate at a temperature of 90°C.
ii. Primer Annealing:- Primers are attached at the 5 'end of single chains at a temperature of 55°C.
iii. Polymerization:- DNA polymerase enzyme polymerize the primers at 70°C temperature.
• Heat stable DNA polymerase:-
Ø Normal DNA polymerase is heat sensitive. It is destroyed due to its deformation at 90°C temperature. Therefore, we cannot use normal DNA polymerase in PCR.
Ø Instead, we use heat stable DNA polymerase in PCR which can tolerate high temperature and does not deform.
Uses of PCR:-
i. The amplification of gene fragments as fast alternative of cloning.
ii. The modification of DNA fragments.
iii. The sensitive detection of pathogenic microorganisms, if desired followed by an accurate genotyping.
iv. DNA analysis of arachaeological specimens.
v. The detection of mutations relevant for inherited diseases, malignant transformation or tissue typing.
vi. The analysis of genetic markers for forensic applications, for paternity testing and for the mapping of hereditary traits.
vii. The species-specific amplification of DNA segments between interspersed-repeat elements.
viii. The study of gene expression.
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Secondary Metabolites:- These are organic compounds produced by any lifeform, e.g. bacteria, fungi, animals, or plants, which are not directly involved in the metabolism for normal growth, development, or reproduction of the organism.
Classification:-
1. Terpenes:-
> These are the largest single class of compounds found in essential oils.
> They are responsible for the aromas, flavors, and colours.
> These are polymer of isoprene molecules. Hence also called as isoprenoids. Each isoprene molecule contains five carbon atoms with double bonds.
> Examples:- Geraniol (Monoterpene), Cafestol (Diterpene), Lycopene (Tetraterpene), Gutta-percha (Polyterpene)
2. Phenolics:-
> Most abundant secondary metabolites of plants.
> They have potent antioxidant properties.
> They are polyphenols which are ubiquitous means found in all plant organs.
> Examples:-
i. Lignin (Structural polymer)
ii. Flavonoids and Carotenoids (Attractants)
iii. Flavonoids (UV screens)
iv. Salicylic acid and Flavonoids (Signal compounds)
v. Tannins and Phytoalexins (Defense response chemicals)
vi. Coumarins
3. Alkaloids:-
> These are N - containing cyclic organic compound.
> The term ‘alkaloid’ was coined by W. Meibner, a German pharmacist, meaning ‘alkali like’. Latter it was demonstrated that the alkalinity was due to the presence of a basic nitrogen atom.
> Morphine was the first alkaloid which was isolated by F.W. Serturner in 1806 from the latex of the opium poppy.
Types of Alkaloids:-
a. Based upon type of nitrogen and the biochemical origin:-
i. True alkaloids:- They are derived from amino acids and have nitrogen in a heterocyclic ring. Eg.- Cocaine, Morphine, Quinine etc.
ii. Proto alkaloids:- They are derived from amino acids and do not have nitrogen in a heterocyclic ring. Eg.- Yohimbine, Mescaline, Hordenine etc.
iii. Pseudo alkaloids:- They are not derived from amino acids but have nitrogen in a heterocyclic ring. Eg.- Capsaicin, Caffeine, Ephedrine etc.
b. Based upon ring structure:-
i. Tropane alkaloids:- They are abundantly found in the Solanaceae family. They are derived from ornithine and acetoacetate. Eg.- Hyoscyamine, Cocaine, Atropine, Scopolamine
ii. Pyrrolizidine alkaloids:- They occur in the plants from Asteraceae and Fabaceae family. Eg.- Senecionine
iii. Piperidine alkaloids:- Presence of odor is the common feature. Eg.- Trigonelline, Coniine, Arecoline, Lobeline, Pelletierine, Nicotine, Anabasine, Piperine and Ricinine.
iv. Quinolines alkaloids:- These alkaloids are obtained exclusively from the bark of the Cinchona plant. Eg.- Cinchonine, Cinchonidine, Quinine, and Quinidine
v. Isoquinoline alkaloids:- These groups of alkaloids have huge types of medicinal properties like antiviral, antifungal, anticancer, antioxidant, antispasmodic, and an enzyme inhibitor. Eg.- Morphine, Codeine, Papaverine, Narcotine, Hydrastine, Berberine, Emetine, Cephaëline, Tubocurarine, Corydaline.
vi. Indole alkaloids:- This is the largest and most interesting alkaloid group derived from tryptophan. Polyhalogenation is a common feature of these alkaloids. Eg.- Ergometrine, Ergotamine, Physostigmine, Ajmaline, Serpentine, Reserpine, Yohimbine, Aspidospermine, Vincablastine, Strychnine, Bruceine.
vii. Steroidal alkaloids:- 1,2-Cyclopentane phenanthrene ring system is the characteristic of this type of alkaloids. Eg.- Solanine, Veratrum, Funtamine, Conessine.
viii. Imidazole alkaloids:- The imidazole ring structure is the characteristic of this type of alkaloids. Eg.- Pilocarpine.
ix. Purine alkaloids:- They contain purine ring. Purine is the nitrogenous base. Eg.- Caffeine, Theobromine, Theophylline.
x. Pyrrolidine alkaloids:- Eg.- Hygrine, Stachydrine, Ficine, Brevicolline.
Stratagies to increase production of secondary metabolites in tissue culture:-
1. Origin of plant tissue:- The genetics of the plants have a high influence on the production of secondary metabolites. For example, high fielding cultivars produce high nicotine content compared to low fielding cultivars.
2. Culture Media:- The composition of the culture medium influences both, biomass production and the synthesis of secondary metabolites. So, ideal conditions are required to be maintained to keep a balance between these two.
3. Growth Regulators:- They have a significant effect on the production of secondary metabolites, as they can either be synergistic or antagonists in their actions. For example, the addition of gibberellic acid to the Taxus cuspidate’s medium increases the taxol production.
4. Carbon source in the medium:- Sucrose is one the most widely used carbon source in the culture media. However, there are also some others, which include glucose, fructose, and galactose.
5. Temperature:- A higher temperature is reported to enhance the production of secondary metabolites.
6. pH:- A perfect pH between 5-6 is required to be maintained for the production of secondary metabolites.
7. Light intensity:- Apart from stimulating the production of secondary metabolites, light intensity also has a role in determining the amount of the production.
8. High-cell Density culture:- A high-density culture increases the production of secondary metabolites. The case is reported in Japonic cells where they have grown up to 759 \ L of cell mass to increase productivity.
9. Elicitation:- Elicitors are compounds of biological origin involved in plant microbe interaction. Elicitors are considered as mediator compounds which induce secondary metabolites formation in cells cultures. Varieties of elicitors have been used for production of secondary metabolites. Elicitation improves the efficiency of Sec. Product accumulation in plant cell culture by:
i. Minimizing up on time
ii. Avoiding change of media
iii. Induction of enzymes involved in biosynthetic pathway
iv. Inducing excretion of metabolites into the medium
Some of the biotic elicitor- induced products
10. Precursor feeding:- Under in vitro circumstances, often the precursors of metabolite die and can’t carry out the reaction. This can be overcome by supplying enough precursors to cells, which can be done by two methods:
a. Add precussors:- By adding precursors in the medium so that their availability is not limited.
b. Prevent resistant precursor analogs:- By preventing resistant precursor analogs and increasing the intracellular levels of precursors for the metabolite production. Eg.-
i. Production of shikonin increased three-fold after the addition of L-Phenylalanine to the medium in Lithospermum species.
ii. Datura sp. cell suspension cultures are supplemented with hydroquinone, in traces, the arbutin synthesis increase considerably.
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Biotechnology Intellectual Property Rights:-
> Biotechnology intellectual property rights are the legal ownership of an interest in a patent, trademark or trade secret. This means that another company cannot use those assets without permission from the company established as the official owner. In health care, intellectual property rights give their owners exclusive use of pharmaceuticals, brand names and more. Intellectual property rights are often the primary driver of value for these companies, particularly in biotech.
Key Points:-
> Intellectual property rights, as they pertain to the Biotechnology sector, concern the legal ownership and exclusive rights to patents, trademarks, and trade secrets.
> Just like with other industries, intellectual property rights allow biotech firms to establish ownership and protect their products from the threat of competitors.
> A company might own the patent to a specific drug and the exclusive right to market it under a certain name, for example, because it holds the intellectual property rights.
Understanding Biotechnology IPRs:- Biotechnology intellectual property rights provide health care companies with a means to protect their claim to and ownership of these assets through common law, state law or federal law. There is some controversy over intellectual property rights in biotechnology. Those in favor argue that they provide a key incentive for developers to innovate because these protections will allow them to be financially rewarded for successful innovations. Those opposed to the strict enforcement of these protections argue that broader sharing of information would reduce prices and increase access to care, especially in developing countries.
Biotechnology IPRs Examples:-
> Here is one example of how intellectual property rights work in the health care industry. Federal protection allows companies to use the ® symbol with a trade name to indicate that it has a registered trademark and that no one else can use that name. More than one company may sell the same chemical compound, which means the same drug, but only one company can legally use the trademarked name to market that drug.
> For example, while many companies sell the antidepressant drug fluoxetine hydrochloride, only Eli Lilly can call it Prozac. Likewise, only Hoffmann-La Roche can use the trademarked name Tamiflu to market a drug called Oseltamivir that is designed to prevent and treat influenza. Trademarks aren’t just used with drugs, however; they’re also used with hospital names, physician practice names, and other entities with distinct branding. This is of major importance to companies in this business environment, where branding, marketing, and image are central components of business operations and strategic positioning. Some studies estimate that pharmaceutical companies spend as much as $30 billion on marketing annually to raise brand awareness for their drugs.
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