Cholera toxin is AB5 multimeric protein complex secreted by the bacterium Vibrio cholerae. CTX is responsible for the watery diarrhea characteristic of cholera infection, it is a member of the Heat-labile enterotoxin family. Cholera toxin was discovered in 1959 by Indian microbiologist Sambhu Nath De The cholera toxin is an oligomeric complex made up of six protein subunits: a single copy of the A subunit, five copies of the B subunit, denoted as AB5. Subunit B binds; the three-dimensional structure of the toxin was determined using X-ray crystallography by Zhang et al. in 1995. The five B subunits—each weighing 11 kDa, form a five-membered ring; the A subunit, 28 kDa, has two important segments. The A1 portion of the chain is a globular enzyme payload that ADP-ribosylates G proteins, while the A2 chain forms an extended alpha helix which sits snugly in the central pore of the B subunit ring; this structure is similar in shape and sequence to the heat-labile enterotoxin secreted by some strains of the Escherichia coli bacterium.
Cholera toxin acts by the following mechanism: First, the B subunit ring of the cholera toxin binds to GM1 gangliosides on the surface of target cells. The B subunit can bind to cells lacking GM1; the toxin most binds to other types of glycans, such as Lewis Y and Lewis X, attached to proteins instead of lipids. Once bound, the entire toxin complex is endocytosed by the cell and the cholera toxin A1 chain is released by the reduction of a disulfide bridge; the endosome is moved to the Golgi apparatus, where the A1 protein is recognized by the endoplasmic reticulum chaperone, protein disulfide isomerase. The A1 chain is unfolded and delivered to the membrane, where Ero1 triggers the release of the A1 protein by oxidation of protein disulfide isomerase complex; as the A1 protein moves from the ER into the cytoplasm by the Sec61 channel, it refolds and avoids deactivation as a result of ubiquitination. CTA1 is free to bind with a human partner protein called ADP-ribosylation factor 6; the CTA1 fragment catalyses ADP-ribosylation of the Gs alpha subunit proteins using NAD.
The ADP-ribosylation causes the Gαs subunit to lose its catalytic activity of GTP hydrolization into GDP + Pi, thus maintaining Gαs in its activated state. Increased Gαs activation leads to increased adenylate cyclase activity, which increases the intracellular concentration of 3',5'-cyclic AMP to more than 100-fold over normal and over-activates cytosolic PKA; these active PKA phosphorylate the cystic fibrosis transmembrane conductance regulator chloride channel proteins, which leads to ATP-mediated efflux of chloride ions and leads to secretion of H2O, Na+, K+, HCO3− into the intestinal lumen. In addition, the entry of Na+ and the entry of water into enterocytes are diminished; the combined effects result in rapid fluid loss from the intestine, up to 2 liters per hour, leading to severe dehydration and other factors associated with cholera, including a rice-water stool. The pertussis toxin produced by Bordetella pertussis acts in a similar manner with the exception that it ADP-ribosylates the Gαi subunit, rendering it unable to inhibit cAMP production.
The gene encoding the cholera toxin is introduced into V. cholerae by horizontal gene transfer. Virulent strains of V. cholerae hold a virus known as a CTXφ Bacteriophage. Because the B subunit appears to be non-toxic, researchers have found a number of applications for it in cell and molecular biology, it is used as a neuronal tracer. Treatment of cultured rodent neural stem cells with cholera toxin induces changes in the localization of the transcription factor Hes3 and increases their numbers. GM1 gangliosides are found in lipid rafts on the cell surface. B subunit complexes labelled with fluorescent tags or subsequently targeted with antibodies can be used to identify rafts. Enterotoxin Ganglioside 1. De SN. Enterotoxicity of bacteria-free culture filtrate of Vibrio cholerae. Nature. 1959. Http://www.ebi.ac.uk/interpro/potm/2005_9/Page1.htm Molecule of the Month Cholera+Toxin at the US National Library of Medicine Medical Subject Headings
The salivary glands in mammals are exocrine glands that produce saliva through a system of ducts. Humans have three paired major salivary glands as well as hundreds of minor salivary glands. Salivary glands can be classified as mucous or seromucous. In serous secretions, the main type of protein secreted is alpha-amylase, an enzyme that breaks down starch into maltose and glucose, whereas in mucous secretions the main protein secreted is mucin, which acts as a lubricant. In humans, between 0.5 and 1.5 litres of saliva are produced every day. The secretion of saliva is mediated by parasympathetic stimulation; the salivary glands are detailed below: The two parotid glands are major salivary glands wrapped around the mandibular ramus in humans. These are largest of the salivary glands, secreting saliva to facilitate mastication and swallowing, amylase to begin the digestion of starches, it is the serous type of gland. It enters the oral cavity via the parotid duct; the glands are located posterior to the mandibular ramus and anterior to the mastoid process of the temporal bone.
They are clinically relevant in dissections of facial nerve branches while exposing the different lobes, since any iatrogenic lesion will result in either loss of action or strength of muscles involved in facial expression. They produce 20% of the total salivary content in the oral cavity. Mumps is a viral infection, caused by infection in the parotid gland; the submandibular glands are a pair of major salivary glands located beneath the lower jaws, superior to the digastric muscles. The secretion produced is a mixture of both serous fluid and mucus, enters the oral cavity via the submandibular duct or Wharton duct. 65-70% of saliva in the oral cavity is produced by the submandibular glands though they are much smaller than the parotid glands. This gland can be felt via palpation of the neck, as it is in the superficial cervical region and feels like a rounded ball, it is located about two fingers above the Adam's apple and about two inches apart under the chin. The sublingual glands are a pair of major salivary glands located inferior to the tongue, anterior to the submandibular glands.
The secretion produced is mucous in nature. Unlike the other two major glands, the ductal system of the sublingual glands does not have intercalated ducts and does not have striated ducts either, so saliva exits directly from 8-20 excretory ducts known as the Rivinus ducts. 5% of saliva entering the oral cavity comes from these glands. There are 800 to 1,000 minor salivary glands located throughout the oral cavity within the submucosa of the oral mucosa in the tissue of the buccal and lingual mucosa, the soft palate, the lateral parts of the hard palate, the floor of the mouth or between muscle fibers of the tongue, they are 1 to 2 mm in diameter and unlike the major glands, they are not encapsulated by connective tissue, only surrounded by it. The gland has a number of acini connected in a tiny lobule. A minor salivary gland may have a common excretory duct with another gland, or may have its own excretory duct, their secretion is mucous in nature and have many functions such as coating the oral cavity with saliva.
Problems with dentures are sometimes associated with minor salivary glands if there is dry mouth present. The minor salivary glands are innervated by facial nerve. Von Ebner's glands are glands found in a trough circling the circumvallate papillae on the dorsal surface of the tongue near the terminal sulcus, they secrete a purely serous fluid. They facilitate the perception of taste through secretion of digestive enzymes and proteins; the arrangement of these glands around the circumvallate papillae provides a continuous flow of fluid over the great number of taste buds lining the sides of the papillae, is important for dissolving the food particles to be tasted. Salivary glands are innervated, either directly or indirectly, by the parasympathetic and sympathetic arms of the autonomic nervous system. Parasympathetic stimulation evokes a copious flow of saliva. Parasympathetic innervation to the salivary glands is carried via cranial nerves; the parotid gland receives its parasympathetic input from the glossopharyngeal nerve via the otic ganglion, while the submandibular and sublingual glands receive their parasympathetic input from the facial nerve via the submandibular ganglion.
These nerves release acetylcholine and substance P, which activate the IP3 and DAG pathways respectively. Direct sympathetic innervation of the salivary glands takes place via preganglionic nerves in the thoracic segments T1-T3 which synapse in the superior cervical ganglion with postganglionic neurons that release norepinephrine, received by β-adrenergic receptors on the acinar and ductal cells of the salivary glands, leading to an increase in cyclic adenosine monophosphate levels and the corresponding increase of saliva secretion. Note that in this regard both parasympathetic and sympathetic stimuli result in an increase in salivary gland secretions; the sympathetic nervous system affects salivary gland secretions indirectly by innervating the blood vessels that supply the glands. The gland is internally divided into lobules. Blood vessels and nerves enter the glands at the hilum and branch out into the lobules. Secretory cells are found in a group, or acinus (pl
G protein-coupled receptor
G protein-coupled receptors known as seven--transmembrane domain receptors, 7TM receptors, heptahelical receptors, serpentine receptor, G protein–linked receptors, constitute a large protein family of receptors that detect molecules outside the cell and activate internal signal transduction pathways and cellular responses. Coupling with G proteins, they are called seven-transmembrane receptors because they pass through the cell membrane seven times. G protein-coupled receptors are found only in eukaryotes, including yeast, choanoflagellates, animals; the ligands that bind and activate these receptors include light-sensitive compounds, pheromones and neurotransmitters, vary in size from small molecules to peptides to large proteins. G protein-coupled receptors are involved in many diseases, are the target of 34% of all modern medicinal drugs. There are two principal signal transduction pathways involving the G protein-coupled receptors: the cAMP signal pathway and the phosphatidylinositol signal pathway.
When a ligand binds to the GPCR it causes a conformational change in the GPCR, which allows it to act as a guanine nucleotide exchange factor. The GPCR can activate an associated G protein by exchanging the GDP bound to the G protein for a GTP; the G protein's α subunit, together with the bound GTP, can dissociate from the β and γ subunits to further affect intracellular signaling proteins or target functional proteins directly depending on the α subunit type. GPCRs are an important drug target and 34% of all Food and Drug Administration approved drugs target 108 members of this family; the global sales volume for these drugs is estimated to be 180 billion US dollars as of 2018. The 2012 Nobel Prize in Chemistry was awarded to Brian Kobilka and Robert Lefkowitz for their work, "crucial for understanding how G protein-coupled receptors function". There have been at least seven other Nobel Prizes awarded for some aspect of G protein–mediated signaling; as of 2012, two of the top ten global best-selling drugs act by targeting G protein-coupled receptors.
The exact size of the GPCR superfamily is unknown, but at least 810 different human genes have been predicted to code for them from genome sequence analysis. Although numerous classification schemes have been proposed, the superfamily was classically divided into three main classes with no detectable shared sequence homology between classes; the largest class by far is class A. Of class A GPCRs, over half of these are predicted to encode olfactory receptors, while the remaining receptors are liganded by known endogenous compounds or are classified as orphan receptors. Despite the lack of sequence homology between classes, all GPCRs have a common structure and mechanism of signal transduction; the large rhodopsin A group has been further subdivided into 19 subgroups. According to the classical A-F system, GPCRs can be grouped into 6 classes based on sequence homology and functional similarity: Class A Class B Class C Class D Class E Class F More an alternative classification system called GRAFS has been proposed for vertebrate GPCRs.
They correspond to classical classes C, A, B2, F, B. An early study based on available DNA sequence suggested that the human genome encodes 750 G protein-coupled receptors, about 350 of which detect hormones, growth factors, other endogenous ligands. 150 of the GPCRs found in the human genome have unknown functions. Some web-servers and bioinformatics prediction methods have been used for predicting the classification of GPCRs according to their amino acid sequence alone, by means of the pseudo amino acid composition approach. GPCRs are involved in a wide variety of physiological processes; some examples of their physiological roles include: The visual sense: The opsins evolved from early GPCRs over 650 million years ago, use a photoisomerization reaction to translate electromagnetic radiation into cellular signals. Rhodopsin, for example, uses the conversion of 11-cis-retinal to all-trans-retinal for this purpose; the gustatory sense: GPCRs in taste cells mediate release of gustducin in response to bitter-, umami- and sweet-tasting substances.
The sense of smell: Receptors of the olfactory epithelium bind odorants and pheromones Behavioral and mood regulation: Receptors in the mammalian brain bind several different neurotransmitters, including serotonin, dopamine, GABA, glutamate Regulation of immune system activity and inflammation: Chemokine receptors bind ligands that mediate intercellular communication between cells of the immune system. GPCRs are involved in immune-modulation and directly involved in suppression of TLR-induced immune responses from T cells. Autonomic nervous system transmission: Both the sympathetic and parasympathetic nervous systems are regulated by GPCR pathways, responsible for control of many automatic functions of the body such as blood pressure, heart rate, digestive processes Cell density sensing: A novel GPCR role in regulating cell density sensing. Homeostasis modulation. Involved in growth and metastasis of some types of tumors. Used in the endocrine syste
Acetylcholine is an organic chemical that functions in the brain and body of many types of animals, including humans, as a neurotransmitter—a chemical message released by nerve cells to send signals to other cells. Its name is derived from its chemical structure: it is an ester of acetic acid and choline. Parts in the body that use or are affected by acetylcholine are referred to as cholinergic. Substances that interfere with acetylcholine activity are called anticholinergics. Acetylcholine is the neurotransmitter used at the neuromuscular junction—in other words, it is the chemical that motor neurons of the nervous system release in order to activate muscles; this property means that drugs that affect cholinergic systems can have dangerous effects ranging from paralysis to convulsions. Acetylcholine is a neurotransmitter in the autonomic nervous system, both as an internal transmitter for the sympathetic nervous system and as the final product released by the parasympathetic nervous system.
Acetylcholine, has been traced in cells of non-neural origins and microbes. Enzymes related to its synthesis and cellular uptake have been traced back to early origins of unicellular eukaryotes; the protist pathogen Acanthamoeba spp. has shown the presence of ACh, which provides growth and proliferative signals via a membrane located M1-muscarinic receptor homolog. In the brain, acetylcholine functions as a neurotransmitter and as a neuromodulator; the brain contains a number of cholinergic areas, each with distinct functions. Because of its muscle-activating function, but because of its functions in the autonomic nervous system and brain, a large number of important drugs exert their effects by altering cholinergic transmission. Numerous venoms and toxins produced by plants and bacteria, as well as chemical nerve agents such as Sarin, cause harm by inactivating or hyperactivating muscles via their influences on the neuromuscular junction. Drugs that act on muscarinic acetylcholine receptors, such as atropine, can be poisonous in large quantities, but in smaller doses they are used to treat certain heart conditions and eye problems.
Scopolamine, which acts on muscarinic receptors in the brain, can cause delirium and amnesia. The addictive qualities of nicotine are derived from its effects on nicotinic acetylcholine receptors in the brain. Acetylcholine is a choline molecule, acetylated at the oxygen atom; because of the presence of a polar, charged ammonium group, acetylcholine does not penetrate lipid membranes. Because of this, when the drug is introduced externally, it remains in the extracellular space and does not pass through the blood–brain barrier. A synonym of this drug is miochol. Acetylcholine is synthesized in certain neurons by the enzyme choline acetyltransferase from the compounds choline and acetyl-CoA. Cholinergic neurons are capable of producing ACh. An example of a central cholinergic area is the nucleus basalis of Meynert in the basal forebrain; the enzyme acetylcholinesterase converts acetylcholine into the inactive metabolites choline and acetate. This enzyme is abundant in the synaptic cleft, its role in clearing free acetylcholine from the synapse is essential for proper muscle function.
Certain neurotoxins work by inhibiting acetylcholinesterase, thus leading to excess acetylcholine at the neuromuscular junction, causing paralysis of the muscles needed for breathing and stopping the beating of the heart. Acetylcholine functions in both the central nervous system and the peripheral nervous system. In the CNS, cholinergic projections from the basal forebrain to the cerebral cortex and hippocampus support the cognitive functions of those target areas. In the PNS, acetylcholine activates muscles and is a major neurotransmitter in the autonomic nervous system. Like many other biologically active substances, acetylcholine exerts its effects by binding to and activating receptors located on the surface of cells. There are two main classes of acetylcholine receptor and muscarinic, they are named for chemicals that can selectively activate each type of receptor without activating the other: muscarine is a compound found in the mushroom Amanita muscaria. Nicotinic acetylcholine receptors are ligand-gated ion channels permeable to sodium and calcium ions.
In other words, they are ion channels embedded in cell membranes, capable of switching from a closed to an open state when acetylcholine binds to them. Nicotinic receptors come in two main types, known as neuronal-type; the muscle-type can be selectively blocked by the neuronal-type by hexamethonium. The main location of muscle-type receptors is on muscle cells. Neuronal-type receptors are located in autonomic ganglia, in the central nervous system. Muscarinic acetylcholine receptors have a more complex mechanism, affect target cells over a longer time frame. In mammals, five subtypes of muscarinic receptors have been identified, labeled M1 through M5. All of them function as G protein-coupled receptors, meaning that they exert their effects via a second messenger system; the M1, M3, M5 subtypes are Gq-coupled. Their effect on target cells is excitatory; the M2 and M4 subtypes are Gi/Go-coupled. Their effect on target cells is inhibitory. Muscarinic acetylcholine receptors are found in both
Autonomic nervous system
The autonomic nervous system the vegetative nervous system, is a division of the peripheral nervous system that supplies smooth muscle and glands, thus influences the function of internal organs. The autonomic nervous system is a control system that acts unconsciously and regulates bodily functions such as the heart rate, respiratory rate, pupillary response and sexual arousal; this system is the primary mechanism in control of the fight-or-flight response. Within the brain, the autonomic nervous system is regulated by the hypothalamus. Autonomic functions include control of respiration, cardiac regulation, vasomotor activity, certain reflex actions such as coughing, sneezing and vomiting; those are subdivided into other areas and are linked to ANS subsystems and nervous systems external to the brain. The hypothalamus, just above the brain stem, acts as an integrator for autonomic functions, receiving ANS regulatory input from the limbic system to do so; the autonomic nervous system has three branches: the sympathetic nervous system, the parasympathetic nervous system and the enteric nervous system.
Some textbooks do not include the enteric nervous system as part of this system. The sympathetic nervous system is considered the "fight or flight" system, while the parasympathetic nervous system is considered the "rest and digest" or "feed and breed" system. In many cases, both of these systems have "opposite" actions where one system activates a physiological response and the other inhibits it. An older simplification of the sympathetic and parasympathetic nervous systems as "excitatory" and "inhibitory" was overturned due to the many exceptions found. A more modern characterization is that the sympathetic nervous system is a "quick response mobilizing system" and the parasympathetic is a "more activated dampening system", but this has exceptions, such as in sexual arousal and orgasm, wherein both play a role. There are excitatory synapses between neurons. A third subsystem of neurons that have been named non-noradrenergic, non-cholinergic transmitters have been described and found to be integral in autonomic function, in particular in the gut and the lungs.
Although the ANS is known as the visceral nervous system, the ANS is only connected with the motor side. Most autonomous functions are involuntary but they can work in conjunction with the somatic nervous system which provides voluntary control; the autonomic nervous system is divided into the sympathetic nervous system and parasympathetic nervous system. The sympathetic division emerges from the spinal cord in the thoracic and lumbar areas, terminating around L2-3; the parasympathetic division has craniosacral “outflow”, meaning that the neurons begin at the cranial nerves and sacral spinal cord. The autonomic nervous system is unique in; the preganglionic, or first, neuron will begin at the “outflow” and will synapse at the postganglionic, or second, neuron's cell body. The postganglionic neuron will synapse at the target organ; the sympathetic nervous system consists of cells with bodies in the lateral grey column from T1 to L2/3. These cell bodies are "GVE" are the preganglionic neurons. There are several locations upon which preganglionic neurons can synapse for their postganglionic neurons: Paravertebral ganglia of the sympathetic chain cervical ganglia thoracic ganglia and rostral lumbar ganglia caudal lumbar ganglia and sacral gangliaPrevertebral ganglia Chromaffin cells of the adrenal medulla These ganglia provide the postganglionic neurons from which innervation of target organs follows.
Examples of splanchnic nerves are: Cervical cardiac nerves & thoracic visceral nerves, which synapse in the sympathetic chain Thoracic splanchnic nerves, which synapse in the prevertebral ganglia Lumbar splanchnic nerves, which synapse in the prevertebral ganglia Sacral splanchnic nerves, which synapse in the inferior hypogastric plexusThese all contain afferent nerves as well, known as GVA neurons. The parasympathetic nervous system consists of cells with bodies in one of two locations: the brainstem or the sacral spinal cord; these are the preganglionic neurons, which synapse with postganglionic neurons in these locations: Parasympathetic ganglia of the head: Ciliary, Submandibular and Otic In or near the wall of an organ innervated by the Vagus or Sacral nerves These ganglia provide the postganglionic neurons from which innervations of target organs follows. Examples are: The postganglionic parasympathetic splanchnic nerves The vagus nerve, which passes through the thorax and abdominal regions innervating, among other organs, the heart, lungs and stomach The sensory arm is composed of primary visceral sensory neurons found in the peripheral nervous system, in cranial sensory ganglia: the geniculate and nodose ganglia, appen
Ligand-gated ion channel
Ligand-gated ion channels commonly referred to as ionotropic receptors, are a group of transmembrane ion-channel proteins which open to allow ions such as Na+, K+, Ca2+, and/or Cl− to pass through the membrane in response to the binding of a chemical messenger, such as a neurotransmitter. When a presynaptic neuron is excited, it releases a neurotransmitter from vesicles into the synaptic cleft; the neurotransmitter binds to receptors located on the postsynaptic neuron. If these receptors are ligand-gated ion channels, a resulting conformational change opens the ion channels, which leads to a flow of ions across the cell membrane. This, in turn, results in either a depolarization, for an excitatory receptor response, or a hyperpolarization, for an inhibitory response; these receptor proteins are composed of at least two different domains: a transmembrane domain which includes the ion pore, an extracellular domain which includes the ligand binding location. This modularity has enabled a ` conquer' approach to finding the structure of the proteins.
The function of such receptors located at synapses is to convert the chemical signal of presynaptically released neurotransmitter directly and quickly into a postsynaptic electrical signal. Many LICs are additionally modulated by allosteric ligands, by channel blockers, ions, or the membrane potential. LICs are classified into three superfamilies which lack evolutionary relationship: cys-loop receptors, ionotropic glutamate receptors and ATP-gated channels; the cys-loop receptors are named after a characteristic loop formed by a disulfide bond between two cysteine residues in the N terminal extracellular domain. They are part of a larger family of pentameric ligand-gated ion channels that lack this disulfide bond, hence the tentative name "Pro-loop receptors". A binding site in the extracellular N-terminal ligand-binding domain gives them receptor specificity for acetylcholine, glycine, glutamate and γ-aminobutyric acid in vertebrates; the receptors are subdivided with respect to the type of ion that they conduct and further into families defined by the endogenous ligand.
They are pentameric with each subunit containing 4 transmembrane helices constituting the transmembrane domain, a beta sheet sandwich type, extracellular, N terminal, ligand binding domain. Some contain an intracellular domain like shown in the image; the prototypic ligand-gated ion channel is the nicotinic acetylcholine receptor. It consists with two binding sites for acetylcholine; when the acetylcholine binds it alters the receptor's configuration and causes the constriction in the pore of 3 angstroms to widen to 8 angstroms so that ions can pass through. This pore allows. With a sufficient number of channels opening at once, the inward flow of positive charges carried by Na+ ions depolarizes the postsynaptic membrane sufficiently to initiate an action potential. While single-cell organisms like bacteria would have little apparent need for the transmission of an action potential, a bacterial homologue to an LIC has been identified, hypothesized to act nonetheless as a chemoreceptor; this prokaryotic nAChR variant is known as the GLIC receptor, after the species in which it was identified.
Cys-loop receptors have structural elements that are well conserved, with a large extracellular domain harboring an alpha-helix and 10 beta-strands. Following the ECD, four transmembrane segments are connected by intracellular and extracellular loop structures. Except the TMS 3-4 loop, their lengths are only 7-14 residues; the TMS 3-4 loop forms the largest part of the intracellular domain and exhibits the most variable region between all of these homologous receptors. The ICD is defined by the TMS 3-4 loop together with the TMS 1-2 loop preceding the ion channel pore. Crystallization has revealed structures for some members of the family, but to allow crystallization, the intracellular loop was replaced by a short linker present in prokaryotic cys-loop receptors, so their structures as not known; this intracellular loop appears to function in desensitization, modulation of channel physiology by pharmacological substances, posttranslational modifications. Motifs important for trafficking are therein, the ICD interacts with scaffold proteins enabling inhibitory synapse formation.
The ionotropic glutamate receptors bind the neurotransmitter glutamate. They form tetramers with each subunit consisting of an extracellular amino terminal domain, an extracellular ligand binding domain, a transmembrane domain; the transmembrane domain of each subunit contains three transmembrane helices as well as a half membrane helix with a reentrant loop. The structure of the protein starts with the ATD at the N terminus followed by the first half of the LBD, interrupted by helices 1,2 and 3 of the TMD before continuing with the final half of the LBD and finishing with helix 4 of the TMD at the C terminus; this means there are three links between the extracellular domains. Each subunit of the tetramer has a binding site for glutamate formed by the two LBD sections forming a clamshell like shape. Only two of these sites in the tetramer need to be occupied to open the ion c
Molecular biology is a branch of biology that concerns the molecular basis of biological activity between biomolecules in the various systems of a cell, including the interactions between DNA, RNA, proteins and their biosynthesis, as well as the regulation of these interactions. Writing in Nature in 1961, William Astbury described molecular biology as:...not so much a technique as an approach, an approach from the viewpoint of the so-called basic sciences with the leading idea of searching below the large-scale manifestations of classical biology for the corresponding molecular plan. It is concerned with the forms of biological molecules and is predominantly three-dimensional and structural – which does not mean, that it is a refinement of morphology, it must at the same time inquire into function. Researchers in molecular biology use specific techniques native to molecular biology but combine these with techniques and ideas from genetics and biochemistry. There is not a defined line between these disciplines.
This is shown in the following schematic that depicts one possible view of the relationships between the fields: Biochemistry is the study of the chemical substances and vital processes occurring in live organisms. Biochemists focus on the role and structure of biomolecules; the study of the chemistry behind biological processes and the synthesis of biologically active molecules are examples of biochemistry. Genetics is the study of the effect of genetic differences in organisms; this can be inferred by the absence of a normal component. The study of "mutants" – organisms which lack one or more functional components with respect to the so-called "wild type" or normal phenotype. Genetic interactions can confound simple interpretations of such "knockout" studies. Molecular biology is the study of molecular underpinnings of the processes of replication, transcription and cell function; the central dogma of molecular biology where genetic material is transcribed into RNA and translated into protein, despite being oversimplified, still provides a good starting point for understanding the field.
The picture has been revised in light of emerging novel roles for RNA. Much of molecular biology is quantitative, much work has been done at its interface with computer science in bioinformatics and computational biology. In the early 2000s, the study of gene structure and function, molecular genetics, has been among the most prominent sub-fields of molecular biology. Many other areas of biology focus on molecules, either directly studying interactions in their own right such as in cell biology and developmental biology, or indirectly, where molecular techniques are used to infer historical attributes of populations or species, as in fields in evolutionary biology such as population genetics and phylogenetics. There is a long tradition of studying biomolecules "from the ground up" in biophysics. One of the most basic techniques of molecular biology to study protein function is molecular cloning. In this technique, DNA coding for a protein of interest is cloned using polymerase chain reaction, and/or restriction enzymes into a plasmid.
A vector has 3 distinctive features: an origin of replication, a multiple cloning site, a selective marker antibiotic resistance. Located upstream of the multiple cloning site are the promoter regions and the transcription start site which regulate the expression of cloned gene; this plasmid can be inserted into either bacterial or animal cells. Introducing DNA into bacterial cells can be done by transformation via uptake of naked DNA, conjugation via cell-cell contact or by transduction via viral vector. Introducing DNA into eukaryotic cells, such as animal cells, by physical or chemical means is called transfection. Several different transfection techniques are available, such as calcium phosphate transfection, electroporation and liposome transfection; the plasmid may be integrated into the genome, resulting in a stable transfection, or may remain independent of the genome, called transient transfection. DNA coding for a protein of interest is now inside a cell, the protein can now be expressed.
A variety of systems, such as inducible promoters and specific cell-signaling factors, are available to help express the protein of interest at high levels. Large quantities of a protein can be extracted from the bacterial or eukaryotic cell; the protein can be tested for enzymatic activity under a variety of situations, the protein may be crystallized so its tertiary structure can be studied, or, in the pharmaceutical industry, the activity of new drugs against the protein can be studied. Polymerase chain reaction is an versatile technique for copying DNA. In brief, PCR allows a specific DNA sequence to be modified in predetermined ways; the reaction is powerful and under perfect conditions could amplify one DNA molecule to become 1.07 billion molecules in less than two hours. The PCR technique can be used to introduce restriction enzyme sites to ends of DNA molecules, or to mutate particular bases of DNA, the latter is a method referred to as site-directed mutagenesis. PCR can be used to determine whether a particular DNA fragment is found in a cDNA library.
PCR has many variations, like reverse transcription PCR for amplification of RNA, more quantitative PCR which allow for quantitative measurement of DNA or RNA molecules. Gel electrophoresis is one of the principal tools of molecular biology; the basic principle is that DNA, RNA, proteins can all be separated by means of an electric field and size. In agarose gel electrophoresis, DNA and RNA can be separated on th