Primosome:- It is a multi-protein complex that synthesizes RNA primers during DNA replication. These RNA primers provide the free 3′-OH group required for DNA polymerases to begin DNA synthesis.
Components of the bacterial primosome:- In bacteria (such as E. coli), the primosome mainly consists of:
Helicase (DnaB):- Unwinds the DNA double helix at the replication fork.
Primase (DnaG):- Synthesizes short RNA primers (typically 10–12 nucleotides long).
Helicase loader (DnaC):- Helps load DnaB onto DNA during initiation (not part of the active primosome after loading).
Functions:-
> Unwinds the parental DNA.
> Synthesizes RNA primers.
> Enables DNA polymerase III to initiate DNA synthesis.
> Particularly important for repeated primer synthesis on the lagging strand during formation of Okazaki fragments.
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Catenated DNA (Interlinked DNA Molecules):- Catenated DNA is a condition in which two or more circular DNA molecules become physically interlocked like links in a chain after DNA replication. The word "catenated" comes from the Latin catena, meaning chain.
Formation of Catenated DNA:-
> During DNA replication of circular DNA (such as bacterial chromosomes and plasmids), the two daughter DNA molecules remain linked together.
> This interlocking occurs because the DNA double helix is twisted around itself.
> The linked DNA molecules are called catenanes.
Removal of Catenation (Decatenation):-
> The process of separating catenated DNA is called decatenation.
> It is carried out by Type II topoisomerases:
i. DNA Topoisomerase IV (mainly in bacteria)
ii. DNA Gyrase (also contributes in bacteria)
iii. Topoisomerase II (in eukaryotes)
> These enzymes temporarily cut both strands of one DNA molecule, pass the other DNA molecule through the break, and then reseal the DNA.
Importance:-
> Ensures proper separation of daughter DNA molecules after replication.
> Essential for accurate chromosome segregation during cell division.
> Prevents DNA entanglement and maintains genome stability.
> Necessary for the inheritance of plasmids and bacterial chromosomes.
Occurrence:-
> Common in bacterial circular chromosomes.
> Found in plasmids.
> Also occurs in mitochondrial and chloroplast DNA of some organisms.
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Cybrids:- When nucleic and cytoplasm from one species and only cytoplasm from another species come during somatic hybrids, the hybrids thus formed are called cybrids.
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Flavr Savr:- It was the first genetically modified (GM) food approved for human consumption. It was developed by Calgene and approved by the U.S. FDA in 1994.
> Its key feature was that it was engineered to ripen more slowly, allowing tomatoes to stay firm longer during shipping while developing better flavor on the vine.
How it worked:- Scientists reduced the activity of the enzyme polygalacturonase, which breaks down pectin in cell walls and causes tomatoes to soften. By suppressing the gene for this enzyme (using antisense RNA technology), the tomatoes:
> Softened more slowly
> Had a longer shelf life
> Could remain on the vine longer before harvest
Why it was important:-
> It demonstrated that genetically engineered foods could be safely commercialized.
> The FDA concluded it was as safe and nutritious as conventional tomatoes.
> It became a milestone in agricultural biotechnology.
Why it disappeared:- Despite its scientific success, the Flavr Savr was not a commercial success because:
> Production costs were high.
> The tomato varieties used were not ideal for large-scale farming.
> The fruit was still somewhat fragile during transport.
> Monsanto acquired Calgene in 1997 and eventually discontinued the product.
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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.
Procedure:-
a. Digestion:- Digest the DNA with an appropriate restriction enzyme.
b. Electrophoresis:- Run the digest on an agarose gel.
c. Denaturation:- Denature the DNA on the gel. For this soak it in about NaOH, which would separate double-stranded DNA into single-stranded DNA. Only ssDNA can transfer.
d. Depurination:- Fragments greater than 15 kb are hard to transfer to the blotting membrane. Depurination with HCl takes the purines out, cutting the DNA into smaller fragments. Neutralize the acid after this step.
e. Blotting:-
- It is the transfer the denatured DNA to the membrane.
- A nitrocellulose membrane or nylon membrane is used. Nitrocellulose typically has a binding capacity of about 100µg/cm, while nylon has a binding capacity of about 500 µg/cm.
- Many scientists feel nylon is better since it binds more and is less fragile.
- Transfer is usually done by capillary action, which takes several hours. Capillary action transfer draws the buffer up by capillary action through the gel an into the membrane, which will bind ssDNA.
- You may use a vacuum blot apparatus instead of capillary action. In this procedure, a vacuum sucks SSC (Saline Sodium Citrate) through the membrane. This works similarly to capillary action, except more SSC goes through the gel and membrane, so it is faster (about an hour). (SSC provides the high salt level that you need to transfer DNA.)
- After you transfer your DNA to the membrane, treat it with UV light. This cross links (via covalent bonds) the DNA to the membrane. (You can also bake nitrocellulose at about 80C for a couple of hours, but be aware that it is very combustible.)
f. Hybridization:-
- It is the process of annealing the probe to the DNA on the membrane due to complementarity.
- Probing is often done with:
i. 32P labeled ATP
ii. Biotin / Streptavidin
iii. Bioluminescent probe
g. Visualization:-
- Visualize your radioactively labeled target sequence.
- If you used a radiolabeled 32P probe, then you would visualize by autoradiograph.
- Biotin / Streptavidin detection is done by colorimetric methods.
- Bioluminescent visualization uses luminesence.
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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.
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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.
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Biotransformation:- It is the conversion of simple, readily available, and inexpensive molecules into expensive and valuable final products by using plant cell cultures (biological systems).
Techniques of Biotransformation:-
1. Immobilized cells:- It has been reported that immobilized plant cells might have a higher production rate under some situations compared to free cells in the suspensions. In this technique, cells are immobilized using a gel of calcium alginate, polyvinyl alcohol resin on fixed support of foam, fabric, or hollow fibers for the accumulation of secondary metabolites.
Examples of biotransformation by immobilized plant-cells
2. Hairy root culture:- It’s one of the most widely used techniques to obtain secondary metabolites. This is because plants’ roots are a rich source of propane alkaloids, Catharanthus alkaloids, atropine, and hyoscyamine. In this method, hairy roots are sub-cultured on a solid medium and then placed on a rotary shaker at 1000 rpm at 250°C in the dark in a liquid medium.
Examples of biotransformation using root cultures
3. Free cells:- The biotransformation in free cells is taken by many reactions including methylation, oxidation, hydroxylation, and acylation. The cells for this purpose are grown mainly using two techniques:
a. Callus Culture:- Callus is an undifferentiated mass of cells. In tissue culture, it occurs when explants are cultured on a medium and the cells undergo differentiation and redifferentiation to develop into a whole plant.
b. Suspension Culture:- In this method, cells are suspended in a liquid medium. This is done by transferring a cell mass into a liquid medium and continuous agitation into an Erlenmeyer flask. The type of suspension cultures include:
i. Batch suspension cultures
ii. Semi-continuous culture
iii. Continuous culture
Examples of biotransformation by plant-cell culture
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
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Process of Gene Splicing (RNA Splicing):- Gene splicing is the process by which introns (non-coding sequences) are removed from the primary RNA transcript (pre-mRNA) and exons (coding sequences) are joined together to form a mature mRNA, which is then translated into a protein.
Steps of Gene Splicing:-
i. Transcription:-
> DNA is transcribed into pre-mRNA (hnRNA).
> The pre-mRNA contains both exons and introns.
ii. Recognition of Splice Sites:-
> The spliceosome recognizes the 5′ splice site, branch point, and 3′ splice site.
> The spliceosome is made of snRNPs (small nuclear ribonucleoproteins) and proteins.
iii. Removal of Introns:-
> The intron is cut at the 5′ splice site.
> It forms a lariat (loop) structure with the branch point adenine.
> The intron is then cut at the 3′ splice site and removed.
iv. Joining of Exons:-
> The adjacent exons are joined together by phosphodiester bonds.
> This produces a continuous coding sequence.
v. Formation of Mature mRNA:-
> The mature mRNA undergoes 5′ capping and 3′ poly-A tail addition.
> It is transported from the nucleus to the cytoplasm for protein synthesis.
Key Components:-
Pre-mRNA (hnRNA):- Initial RNA transcript
Introns:- Non-coding sequences removed during splicing
Exons:- Coding sequences retained
Spliceosome:- RNA-protein complex that performs splicing
snRNPs (U1, U2, U4, U5, U6):- Components of the spliceosome
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Indirect Gene Transfer:- When stable transformation is achieved by incorporating genes into the genome of plant cells with the help of a biological factor, it is called indirect gene transfer. Agrobacterium bacterium is used in this.
Agrobacterium mediated Gene Transfer:-
• Agrobacterium is a gram -ve bacterium found in soil around plant roots.
• Two species of Agrobacterium bacteria cause diseases in dicot plants:-
i. Agrobacterium tumefaciens
ii. Agrobacterium rhizogenes
i. Agrobacterium tumefaciens:- It causes Crown gall disease in angiosperm plants. Ti-plasmid is found in it.
ii. Agrobacterium rhizogenes:- It cause hairy root disease in angiosperm plants. Ri - plasmid is found in it.
• Recombinant DNA is made by incorporating the desired gene into the T - DNA portion of the Ti plasmid. Which are transferred to Agrobacterium cell.
• Now cut round pieces of sterilized leaf.
• Now incubate these pieces with Agrobacterium overnight.
• Now kept these pieces in Shooting medium for 2 days.
• Now add Kanamycin and Carbenicillin in this shooting medium and cultured for 3-4 weeks.
• Now transfer this to the rooting medium, add Kanamycin and Carbenicilin, culture it for 3-4 weeks.
• Now establish this plant in a pot in green house.
• After a few days, establish the plant in the soil of the field.
Mechanism of Gene Transfer:-
• The T - DNA portion of the Ti - plasmid has the property that it can integrate into the plant's genome.
• Leaf pieces release the Aceto - Syringine chemical that activates the Vir - Operon of T - DNA.
• Once activated, the T-DNA portion separates from the plasmid and enters into many plant cells.
• Now this T-DNA integrates into the genome by going into the nucleus.
• This results in stable transformation of the plant.
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