2021 Solved Old Paper (BOT - 402) New OK

Okazaki fragments:- These are short segments of newly synthesized DNA that are produced on the lagging strand during DNA replication.
Why are they formed?:-
> DNA polymerase can only synthesize DNA in the 5′ → 3′ direction.
Leading strand:- Synthesized continuously toward the replication fork.
Lagging strand:- Synthesized discontinuously away from the replication fork as short pieces called Okazaki fragments.
Size:-
Prokaryotes:- Approximately 1,000–2,000 nucleotides long.
Eukaryotes:- Approximately 100–200 nucleotides long.
Attenuation:- Attenuation is a regulatory mechanism used in bacterial operons to ensure proper transcription and translation. In bacteria, transcription and translation are capable of proceeding simultaneously. The need to prevent unregulated and unnecessary gene expression can be prevented by attenuation, which is characterized as a regulatory mechanism.
i. Transcriptional-attenuation:- It is characterized by the presence of an attenuator within the DNA sequence that results in formation of mRNA-stem loops that prevent further transcription from occurring. The non-functional RNA produced prevents proper transcription.
ii. Translational-attenuation:- It is characterized by the misfolding of the Shine-Dalgarno sequence. The Shine-Dalgarno sequence, responsible for ribosomal binding to allow proper translation, is inaccessible because it is folded into a hairpin-loop structure, thus, translation cannot occur.
Sources of Genetic Variation:- The main sources of genetic variation are:
i. Mutation:- Permanent changes in the DNA sequence that create new alleles. It is the ultimate source of new genetic variation.
ii. Genetic recombination (crossing over):- During meiosis, homologous chromosomes exchange segments, producing new combinations of genes.
iii. Independent assortment:- During meiosis, chromosomes are distributed randomly into gametes, creating different combinations of maternal and paternal chromosomes.
iv. Random fertilization:- Any sperm can fertilize any egg, resulting in unique combinations of genes in offspring.
v. Gene flow (migration):- Movement of individuals or their gametes between populations introduces new alleles into a population.
vi. Horizontal gene transfer:- Common in bacteria, where genes are transferred directly between organisms through processes such as transformation, transduction, and conjugation.

Operon Concept:-

·  “Each metabolic reaction is controlled by a set of genes”

·  All the genes regulating a metabolic reaction constitute an Operon. E.g. lac operon, trp operon, ara operon, his operon, val operon etc.

·  When a substrate is added to growth medium of bacteria, a set of genes is switched on to metabolize it. This is called induction.

·  When a metabolite (product) is added, the genes to produce it are turned off. This is called repression.

Role of Vectors in Recombinant DNA Technology (RDT):- A vector is a DNA molecule used to carry a foreign DNA fragment (gene of interest) into a host cell, where it can replicate and/or express the inserted gene.
Role (Functions) of Vectors in RDT:-
> Carries foreign DNA into the host cell.
> Allows replication of the inserted DNA.
> Enables cloning of the gene of interest.
> Facilitates expression of the inserted gene.
> Contains selectable marker genes (e.g., antibiotic resistance genes) to identify transformed cells.
> Provides restriction sites (Multiple Cloning Site, MCS) for insertion of foreign DNA.
> Maintains stability of recombinant DNA inside the host cell.
> Helps in large-scale production of recombinant proteins and useful biomolecules.

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

Evidences in favor of semi conservative replication:- Matthew Messelson & Franklin Stahl (1958) experimentally proved Semi-conservative model.
> They cultured E. coli in a medium containing 15NH4Cl (15N: heavy isotope of N). 15N was incorporated into both strands of bacterial DNA and the DNA became heavier.
> Another preparation containing N salts labeled with 14N is also made. 14N was also incorporated in both strands of DNA and became lighter.
> These 2 types of DNA can be separated by centrifugation in a CsCl density gradient.
> They took E. coli cells from 15N medium and transferred to 14N medium.
> After one generation (i.e. after 20 minutes), they isolated and centrifuged the DNA. Its density was intermediate (hybrid) between 15N DNA and 14N DNA. This shows that the newly formed DNA one strand is old (15N type) and one strand is new (14N type). This confirms semi-conservative replication.
> After II generation (i.e. after 40 minutes), there was equal amounts of hybrid DNA and light DNA.
A, B and Z – DNA:- 
Evolution of the Gene Concept: From Mendel to the Molecular Level:- The concept of the gene has evolved significantly from an abstract unit of heredity to a well-defined molecular entity.
Flow Chart:-
Mendel's Factors (1866)
⬇
Genes on Chromosomes (Sutton & Boveri, 1902)
⬇
Gene Term Coined (Johannsen, 1909)
⬇
Linear Arrangement of Genes (Morgan, 1910)
⬇
One Gene–One Enzyme (Beadle & Tatum, 1941)
⬇
DNA is Genetic Material & Double Helix (Watson & Crick, 1953)
⬇
Fine Structure of Gene (Benzer, 1955–57)
⬇
One Gene–One Polypeptide (Crick, 1958)
⬇
Regulatory Genes (Jacob & Monod, 1961)
⬇
Modern Molecular Gene: DNA sequence encoding functional RNA/protein with regulatory elements
Biotechnology:- Biotechnology is the branch of science and technology that uses living organisms, cells, enzymes, or biological systems to develop useful products and processes for the benefit of humans.
Definition by EFB (European Federation of Biotechnology):- "Biotechnology is the integration of natural science and engineering to achieve the application of organisms, cells, parts thereof, and molecular analogues for products and services."
Concept of Biotechnology:- Biotechnology combines biology, chemistry, genetics, microbiology, molecular biology, biochemistry, engineering, and computer science to improve living organisms or produce valuable products.
Key Concepts:-
> Utilizes living organisms or their components.
> Involves genetic modification and molecular techniques.
> Produces improved plants, animals, microorganisms, and medicines.
> Helps solve problems in agriculture, medicine, industry, and the environment.
> Integrates traditional fermentation with modern genetic engineering.
Scope of Biotechnology:- Biotechnology has a very wide scope in different sectors.
1. Agricultural Biotechnology:-
> Development of high-yielding crop varieties.
> Production of disease- and pest-resistant crops.
> Drought- and salinity-tolerant crops.
> Tissue culture and micropropagation.
> Biofertilizers and biopesticides.
> Marker-assisted breeding.
2. Medical Biotechnology:-
> Production of vaccines.
> Recombinant insulin and hormones.
> Gene therapy.
> Stem cell therapy.
> Molecular diagnosis (PCR, ELISA).
> Monoclonal antibodies.
3. Industrial Biotechnology:-
> Production of enzymes.
> Fermentation technology.
> Antibiotics and organic acids.
> Biofuels (bioethanol, biodiesel, biogas).
> Bioplastics and biodegradable materials.
4. Environmental Biotechnology:-
> Bioremediation of polluted soil and water.
> Wastewater treatment.
> Composting.
> Solid waste management.
> Pollution control.
5. Animal Biotechnology:-
> Embryo transfer technology.
> Artificial insemination.
> Animal cloning.
> Production of transgenic animals.
> Improvement of livestock breeds.
6. Food Biotechnology:-
> Fermented food products.
> Nutritional enhancement of foods.
> Food preservation.
> Probiotics production.
> Quality testing and food safety.
7. Marine Biotechnology:-
> Production of pharmaceuticals from marine organisms.
> Aquaculture improvement.
> Marine bioactive compounds.
8. Forensic Biotechnology:-
> DNA fingerprinting.
> Crime investigation.
> Paternity testing.
> Identification of individuals.
Somatic Hybridization:- The process of obtaining hybrid plants by fusion of protoplasts of two different species or varieties, is called somatic hybridization. It has 4 main steps -
1. Protoplast Isolation
2. Protoplast Fusion
3. Selection of Hybrid Cells
4. Culturing of Hybrid Cells
1. Protoplast Isolation:- It has 2 main steps -
a. Sterilization of Leaf
b. Enzyme Treatment
a. Sterilization of Leaf:- Soak the leaf in 70% ethanol for 1 minute. After this, keep this leaf in 2% NaOCl solution for 20 to 30 minutes. Now wash this leaf with distilled water for 3 minutes.
b. Enzyme Treatment:- Now this sterilized leaf is treated with 2 enzymes, pectinase and cellulase, respectively. The pectinase enzyme disintegrates the middle lamella. Cellulase enzymes decompose the cell wall. As a result protoplasts are obtained. To increase osmotic concentration, add 500 - 800 ml / L of sorbitol or mannitol.
2. Protoplast Fusion:- For this, 2 different protoplast samples are mixed together. Now Fusogen is added to this mixture which induces protoplast fusion.
One of the following factors can be used as Fusogen -
i. PEG (Poly Ethylene Glycol)
ii. High pH + Ca2+
iii. NaNO3
iv. Electric Pulse
At the end of this step, 3 types of products are obtained -
i. Homokaryon
ii. Heterokaryon
iii. Unfused protoplasts
3. Selection of Hybrid Cells:- It has several methods -
i. Visual markers
ii. Staining of protoplast with fluorescent dyes
iii. Culture, Regeneration and Identification
iv. Red anthocynin marker and green protoplast marker:-
Tobacco (green)  X   Carrot (red)
v. Selective markers:-
Grape   X    Soybean
4. Culturing of Hybrid Cells:-
Ø  Isolated hybrid protoplast cells are proliferated on solid MS-medium in patridishes.
Ø  Osmaticum:- It is a substance that acts to increase the osmotic concentration. It is added to both enzyme mixture and culture medium. The protoplast requires osmotic protection during isolation and culture because the cell wall has not yet been formed. One of the following can be used as osmaticum -
i. Sorbitol
ii. Manitol
iii. Glucose
iv. Sucrose
Ø  Cover the patridish with a lid and seal it with paraffin wax so that the medium does not dry at 40ºC.
Ø  Now transfer this patridish to the incubator where the temperature is 25 - 28ºC and the light intensity is 2300 lux.
Ø  Cell wall formation starts after a few hours. The first cell division occurs after 2–7 days.
Ø  Multicellular groups are formed after 2–3 weeks.
Ø  Now these cellular groups are transferred to a medium free of osmaticum so that callus or embryo can develop.
Ø  Further differentiation leads to organogenesis and development of hybrid plants.
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.

Principles of Genetic Engineering:-

1. Introduction:-

> The technology of recombinant DNA was developed in 1973 by Boyer and Cohen.

> It is popularly known as genetic engineering. 

> Recombinat DNA:- When foreign gene is inserted into a vector, then it is called as recombinant DNA.

> Objective:- This is the natural mathod of amplification of gene of interest.


2. Process of Recombinant DNA Technology:- The complete process of recombinant DNA technology includes multiple steps-

Step-1. Isolation of Genetic Material:- The first and the initial step in Recombinant DNA technology is to isolate the desired DNA in its pure form i.e. free from other macromolecules.

Step-2. Cutting the gene at the recognition sites:- The restriction enzymes play a major role in determining the location at which the desired gene is inserted into the vector genome. These reactions are called ‘restriction enzyme digestions’.

Step-3. Ligation of DNA Molecules:- In this step of Ligation, the joining of the two pieces – a cut fragment of DNA and the vector together with the help of the enzyme DNA ligase.

Step-4. Insertion of Recombinant DNA Into Host:- In this step, the recombinant DNA is introduced into a recipient host cell. This process is termed as Transformation. 

Step-5. Amplifying the gene copies:- Once the recombinant DNA is inserted into the host cell, it gets multiplied. As a result the inserted gene of interest is also multiplied.