Recombinant DNA, Chemiosmosis, and Electron Transport Chain

Constructing Recombinant DNA

The following steps are involved in the construction of recombinant DNA:

1. Preparation of the Gene: Gene cloning in bacteria is achieved by cleaving DNA with enzymes called restriction endonucleases, which create small fragments. Each fragment often has a "sticky end." Since eukaryotic genes contain introns that are not processed in bacteria, the DNA for cloning is usually obtained from relevant mRNA through the process of reverse transcription. In cases where nucleotide or amino acid sequences are known, synthetic DNA may also be produced.
2. Insertion into a Vector: The vector is a vehicle, such as a plasmid or bacteriophage, used to transfer DNA into a host cell. The vector is cut with the same restriction endonuclease used to generate the chromosomal DNA fragments. The chromosomal fragments and the linearized vector are incubated with DNA ligase, which joins the DNA molecules. Plasmids containing an inserted fragment are called recombinant plasmids.
3. Transformation of the Host Cell: The mixture of ligated plasmids is introduced into bacterial cells, which take up the DNA through a process called transformation. Transformation is generally carried out by placing actively growing bacterial cells in a cold, dilute solution of CaCl₂, which enhances their ability to take up foreign DNA. E. coli is often used as a host because its molecular biology is well understood. E. coli can transcribe and translate most Gram-positive and Gram-negative genes, with some exceptions like those from actinomycetes.
4. Detection of the Cloned Gene: DNA probes are used to detect recombinants. In a DNA molecule, the two strands are held together by hydrogen bonds. If two similar DNA pieces are mixed and heated, the hydrogen bonds break, and the strands separate. Upon lowering the temperature, the hydrogen bonds reform, and some of the resulting DNA molecules will be hybrids. This concept of DNA hybridization is used to create DNA probes. The transformed colonies are replica-plated onto a nitrocellulose filter and are lysed to release their DNA. The DNA is denatured by heating and fixed to the filter, creating a "DNA print" that corresponds exactly to the position of the colonies on the original plate. This DNA print is then hybridized with a radioactively labeled probe. After washing off unhybridized DNA, the positions of the radioactive spots on the filter are located by autoradiography to identify the colonies containing the required DNA.

Chemiosmotic Theory of Oxidative Phosphorylation

This theory was proposed by Peter Mitchell in 1961. The main features of this theory are as follows:

1. Three key processes occur across the inner mitochondrial membrane: electron transport, proton translocation, and ATP synthesis.
2. The inner mitochondrial membrane contains the Electron Transport System (ETS), and phosphorylation occurs in the headpiece of the ATP synthase complex.
3. ATP formation and electron transport are coupled through proton translocation.
4. At certain stages of the ETS, hydrogen ions (protons) are liberated.
5. Hydrogen ion uptake reactions occur on the matrix side (inside) of the inner membrane, while hydrogen ion liberating reactions occur on the intermembrane space (outer) side.
6. The transfer of electrons occurs from carriers of low redox potential to carriers of high redox potential. This pumps protons from the mitochondrial matrix to the intermembrane space.
7. This movement of protons generates an electrochemical gradient, also known as the proton-motive force, across the inner mitochondrial membrane.
8. Due to this proton gradient, protons flow back into the matrix through a proton channel located in the F₀ part of the F₀F₁ ATP synthase enzyme.
9. This flow of protons activates the ATP synthase enzyme, located in the head of the F₁ particle.
10. ATP synthase catalyzes the synthesis of ATP from ADP and inorganic phosphate, utilizing the energy from the proton gradient.

The Electron Transport Chain (ETC)

The pairs of hydrogen atoms removed from respiratory intermediates during dehydrogenation do not react directly with oxygen. Instead, they are used to reduce hydrogen acceptor molecules, such as NAD⁺ and FAD, to NADH and FADH₂, respectively. These reduced coenzymes release protons (H⁺) into the mitochondrial matrix and channel electrons (e⁻) into the electron transport chain (ETC).

The ETC is a series of electron carriers located in a specific sequence on the inner mitochondrial membrane. These include:

1. Nicotinamide adenine dinucleotide (NAD)
2. Flavin mononucleotide (FMN)
3. Coenzyme Q (or ubiquinone)
4. Cytochrome b
5. Cytochrome c
6. Cytochrome a
7. Cytochrome a₃

The transport of electrons through these carriers is a downhill journey, as electrons flow from a more electronegative to a more electropositive system. This results in a decrease in free energy, with some energy being released at each step. During this electron transfer, the electron donor is oxidized while the electron acceptor is reduced; these are known as redox reactions and are catalyzed by reductases.

At certain steps where a large amount of energy is released, ATP is formed by the phosphorylation of ADP. This process is catalyzed by the enzyme ATP synthase, located at the tip of the F₁ particles. The ETC is therefore also called the oxidative phosphorylation pathway.

Finally, from cytochrome a₃, two electrons are received by an oxygen atom, which also accepts two protons from the mitochondrial matrix to form a water molecule. The reaction is: 2H⁺ + 2e⁻ + ½O₂ → H₂O. This overall reaction is achieved through the many steps of the ETC.

The pairs of hydrogen atoms from respiratory intermediates are received by NAD⁺ or FAD coenzymes. These reduced coenzymes pass their electrons to the ETC. The common electron carrier for both routes is ubiquinone (Coenzyme Q). In Route I, NADH passes its electrons to CoQ via FMN. In Route II, FADH₂ passes its electrons directly to CoQ. The coenzymes then pass these electrons down the cytochrome chain.