Researchers in molecular biology use specific techniques native to molecular biology , but increasingly combine these with techniques and ideas from genetics and biochemistry. There is not a defined line between these disciplines.
The following figure is a schematic that depicts one possible view of the relationship between the fields:
Biochemistry is the study of the chemical substances and vital processes occurring in living organisms. Biochemists focus heavily on the role, function, 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 on organisms. Often this can be inferred by the absence of a normal component ( one gene). 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 (epistasis) can often confound simple interpretations of such "knock-out" studies.
Molecular biology is the study of molecular underpinnings of the process of replication, transcription and translation of the genetic material. The central dogma of molecular biology where genetic material is transcribed into RNA and then translated into protein, despite being an oversimplified picture of molecular biology, still provides a good starting point for understanding the field. This picture, however, is undergoing revision in light of emerging novel roles for RNA.
GC-content (or guanine-cytosine content), in molecular biology, is the percentage of nitrogenous bases on a DNA molecule which are either guanine or cytosine (from a possibility of four different ones, also including adenine and thymine). This may refer to a specific fragment of DNA or RNA, or that of the whole genome. When it refers to a fragment of the genetic material, it may denote the GC-content of part of a gene (domain), single gene, group of genes (or gene clusters) or even a non-coding region. G (guanine) and C (cytosine) undergo a specific hydrogen bonding whereas A (adenine) bonds specifically with T (thymine). The GC pair is bound by three hydrogen bonds and AT paired by two hydrogen bonds, and thus GC pairs are more thermostable compared to the AT pairs. In spite of the higher thermostability conferred to the genetic material, it is envisaged that cells with high GC DNA undergo autolysis, thereby reducing the longitivity of the cell per se.Due to the robustness endowed to the genetic materials in high GC organisms it was commonly believed that the GC content played a vital part in adaptation temperatures, an hypothesis which has recently been refuted.
In PCR experiments, the GC-content of primers are used to determine their annealing temperature to the template DNA. A higher GC-content level indicates a higher melting temperature.
In cell culture, the passaging is the process of sub-culturing animal cells. It is usually done to produce large number of cells from pre-existing ones. Instances where it is followed include vaccine production labs and clonal expansion.
In the average lab, adherent (sticky) mammalian cells are grown in a 10cm-diameter petri dish (a plate), with 10ml of FBS + DMEM media (pink liquid food for cells), in an incubator at 37C with 5% CO2 and a tray of water in the bottom for humidity. In the case of RAW 264.3 or HeLa cells, a 10%-full (10% confluent) plate will reach 100% confluency in two or three days. If nothing is done, the food will run out and the cells will die shortly thereafter, so passaging is required. This is where the media is removed, the cells are washed with PBS (salt water), then 1ml of trypsin is added to make the cells unstick from the botton of the plate. Trypsin works best in the incubator, so the plate is incubated for five minutes. The plate is removed from the incubator, 9ml of PBS is added and the plate is mixed with a pipettor (triturated). An appropriate number of cells in suspension is then transferred new plates, fresh DMEM is added to each plate, the new plates are put in the incubator, and the cycle begins again.
An electron microscope is a type of microscope that uses electrons to illuminate a specimen and create an enlarged image. Electron microscopes have much greater resolving power than light microscopes and can obtain much higher magnifications. Some electron microscopes can magnify specimens up to 2 million times, while the best light microscopes are limited to magnifications of 2000 times. Both electron and light microscopes have resolution limitations, imposed by their wavelength. The greater resolution and magnification of the electron microscope is due to the wavelength of an electron, its de Broglie wavelength, being much smaller than that of a light photon, electromagnetic radiation.
The electron microscope uses electrostatic and electromagnetic lenses in forming the image by controlling the electron beam to focus it at a specific plane relative to the specimen in a manner similar to how a light microscope uses glass lenses to focus light on or through a specimen to form an image.
Electrophoresis is the most known electrokinetic phenomena. It was discovered by Reuss in 1809. He observed that clay particles dispersed in water migrate under influence of an applied electric field. There are detailed descriptions of Electrophoresis in many books on Colloid and Interface Science.There is an IUPAC Technical Report prepared by a group of most known world experts on the electrokinetic phenomena.
Generally, electrophoresis is the motion of dispersed particles relative to a fluid under the influence of an electric field that is space uniform. Alternatively, similar motion in a space non-uniform electric field is called dielectrophoresis.
Electrophoresis occurs because particles dispersed in a fluid almost always carry an electric surface charge. An electric field exerts electrostatic Coulomb force on the particles through these charges. Recent molecular dynamics simulations, though, suggest that surface charge is not always necessary for electrophoresis and that even neutral particles can show electrophoresis due to the specific molecular structure of water at the interface.
The electrostatic Coulomb force exerted on a surface charge is reduced by an opposing force which is electrostatic as well. According to double layer theory, all surface charges in fluids are screened by a diffuse layer. This diffuse layer has the same absolute charge value, but with opposite sign from the surface charge. The electric field induces force on the diffuse layer, as well as on the surface charge. The total value of this force equals to the first mentioned force, but it is oppositely directed. However, only part of this force is applied to the particle. It is actually applied to the ions in the diffuse layer. These ions are at some distance from the particle surface. They transfer part of this electrostatic force to the particle surface through viscous stress. This part of the force that is applied to the particle body is called electrophoretic retardation force.
Gene expression is the process by which inheritable information from a gene, such as the DNA sequence, is made into a functional gene product, such as protein or RNA.
Several steps in the gene expression process may be modulated, including the transcription step and the post-translational modification of a protein. Gene regulation gives the cell control over structure and function, and is the basis for cellular differentiation, morphogenesis and the versatility and adaptability of any organism. Gene regulation may also serve as a substrate for evolutionary change, since control of the timing, location, and amount of gene expression can have a profound effect on the functions (actions) of the gene in the organism.
Post-translational modification (PTM) is the chemical modification of a protein after its translation. It is one of the later steps in protein biosynthesis for many proteins.
The bottom of this diagram shows the modification of primary structure of insulin, as described.
A protein (also called a polypeptide) is a chain of amino acids. During protein synthesis, 20 different amino acids can be incorporated in proteins. After translation, the posttranslational modification of amino acids extends the range of functions of the protein by attaching to it other biochemical functional groups such as acetate, phosphate, various lipids and carbohydrates, by changing the chemical nature of an amino acid (citrullination) or by making structural changes, like the formation of disulfide bridges.
Also, enzymes may remove amino acids from the amino end of the protein, or cut the peptide chain in the middle. For instance, the peptide hormone insulin is cut twice after disulfide bonds are formed, and a propeptide is removed from the middle of the chain; the resulting protein consists of two polypeptide chains connected by disulfide bonds.
Other modifications, like phosphorylation, are part of common mechanisms for controlling the behavior of a protein, for instance activating or inactivating an enzyme.
In the field of molecular biology, a transcription factor (sometimes called a sequence-specific DNA binding factor) is a protein that binds to specific parts of DNA using DNA binding domains and is part of the system that controls the transfer (or transcription) of genetic information from DNA to RNA.
Transcription factors perform this function alone, or by using other proteins in a complex, by increasing (as an activator), or preventing (as a repressor) the presence of RNA polymerase, the enzyme which activates the transcription of genetic information from DNA to RNA.
IMAGE cDNA clones are a collection of DNA vectors containing cDNAs from various organisms including human, mouse, rat, non-human primates, zebrafish, pufferfish, Xenopus (frogs), and cow. Together they represent a more or less complete set of expressed genes from these organisms. IMAGE stands for integrated molecular analysis of genomes and their expression.
An expressed sequence tag or EST is a short sub-sequence of a transcribed spliced nucleotide sequence (either protein-coding or not). They may be used to identify gene transcripts, and are instrumental in gene discovery and gene sequence determination. The identification of ESTs has proceeded rapidly, with approximately 43 million ESTs now available in public databases (e.g. GenBank 6/2007, all species).
An EST is produced by one-shot sequencing of a cloned mRNA (i.e. sequencing several hundred base pairs from an end of a cDNA clone taken from a cDNA library). The resulting sequence is a relatively low quality fragment whose length is limited by current technology to approximately 500 to 800 nucleotides. Because these clones consist of DNA that is complementary to mRNA, the ESTs represent portions of expressed genes. They may be present in the database as either cDNA/mRNA sequence or as the reverse complement of the mRNA, the template strand.
ESTs can be mapped to specific chromosome locations using physical mapping techniques, such as radiation hybrid mapping or FISH. Alternatively, if the genome of the organism that originated the EST has been sequenced one can align the EST sequence to that genome.
The current understanding of the human set of genes (2006) includes the existence of thousands of genes based solely on EST evidence. In this respect, ESTs become a tool to refine the predicted transcripts for those genes, which leads to prediction of their protein products, and eventually of their function. Moreover, the situation in which those ESTs are obtained (tissue, organ, disease state - e.g. cancer) gives information on the conditions in which the corresponding gene is acting. ESTs contain enough information to permit the design of precise probes for DNA microarrays that then can be used to determine the gene expression.
The mucous membranes (or mucosae; singular: mucosa) are linings of mostly endodermal origin, covered in epithelium, which are involved in absorption and secretion. They line various body cavities that are exposed to the external environment and internal organs. It is at several places continuous with skin: at the nostrils, the lips, the ears, the genital area, and the anus. The sticky, thick fluid secreted by the mucous membranes and gland is termed mucus. The term mucous membrane refers to where they are found in the body and not every mucous membrane secretes mucus.
Body cavities featuring mucous membrane include most of the respiratory system. The glans penis (head of the penis) and glans clitoridis and the inside of the prepuce (foreskin) and clitoral hood are mucous membranes, not skin. The secreted mucus traps the pathogens in the body, preventing any further activities of diseases.
An S-layer (surface layer) is a part of the cell envelope commonly found in bacteria, as well as among archaea. It consists of a monomolecular layer composed of identical proteins or glycoproteins. This two dimensional structure is built via self-assembly and encloses the whole cell surface. Thus, the S-layer protein can represent up to 10-15% of the whole protein content of a cell . S-layer proteins are poorly or not conserved at all and can differ markedly even between related species. Depending on species the S-layers have a thickness between 5 and 25 nm and possess identical pores with 2-8 nm in diameter . S-layers exhibit either an oblique (p1, p2), square (p4) or hexagonal (p3, p6) lattice symmetry. Depending on the lattice symmetry the S-layer is composed of one (P1), two (P2), three (P3), four (P4) or six (P6) identical protein subunits, respectively. The centre to centre spacings (or unit cell dimensions) between these subunits range between 2.5 and 35 nm.
Fixation of S-layers in the cell wall
In Gram-negative bacteria S-layers are associated to the LPS via ionic, carbohydrate-carbohydrate, protein carbohydrate interactions and/or protein-protein interactions.
In Gram-positive bacteria whose S-layers contain surface layer homology (SLH) domains the binding occurs to the peptidoglycan and to a secondary cell wall polymer (e.g. teichuronic acids). In the absence of SLH domains the binding occurs via electrostatic interactions between the positively charged N-terminus of the S-layer protein and a negatively charged secondary cell wall polymer.
In Gram-negative archaea S-layer proteins possess a hydrophobic anchor that is associated with the underlying lipid membrane.
In Gram-positive archaea the S-layer proteins bind pseudomurein or to methanochondritin.
As for many bacteria the S-layer represents the outermost interaction zone with their respective environment, its functions are very diverse and vary from species to species. In Gram-negative archaea the S-layer is the only cell wall component and therefore is important for mechanical stabilisation. Additional functions associated with S-layers include:
The endomembrane system is the system of internal membranes within eukaryotic cells that divide the cell into functional and structural compartments, or organelles. Prokaryotes do not have an endomembrane system and thus lack most organelles.
The endomembrane system also provides a transport system, for moving molecules through the interior of the cell, as well as interactive surfaces for lipid and protein synthesis. The membranes that make up the endomembrane system are made of a lipid bilayer, with proteins attached to either side or traversing them.
The following organelles are part of the endomembrane system:
A biofilm is a complex aggregation of microorganisms marked by the excretion of a protective and adhesive matrix. Biofilms are also often characterized by surface attachment, structural heterogeneity, genetic diversity, complex community interactions, and an extracellular matrix of polymeric substances.
Single-celled organisms generally exhibit two distinct modes of behavior. The first is the familiar free floating, or planktonic, form in which single cells float or swim independently in some liquid medium. The second is an attached state in which cells are closely packed and firmly attached to each other and usually form a solid surface. A change in behavior is triggered by many factors, including quorum sensing, as well as other mechanisms that vary between species. When a cell switches modes, it undergoes a phenotypic shift in behavior in which large suites of genes are up- and down- regulated.
Heterogeneous is an adjective used to describe something that has a large amount of variants or different forms. Derived from the Greek; heteros or 'other' and genos or 'kind'.. It is the antonym of homogeneous, which means that an object or system consists of many identical items. Matters of a quantum can exist in homogenous or in heterogeneous or in combined distributions. The term is often used in a scientific (such as a kind of catalyst), mathematical, sociological or statistical context.
A heterogeneous compound, mixture, reaction or other such object is one that consists of many different items, which are often not easily sorted or separated, though they are clearly distinct.
A heterogeneous mixture is a mixture of two or more compounds. In chemical kinetics, a heterogeneous reaction is one that takes place at the interface of two or more i.e. between a solid and a gas, a liquid and a gas, or a solid and a liquid.
amorphous
heterozygous
heteroazeotrope
homogenization
homozygous
mesoporous silicates
Plankton are any drifting organism that inhabits the pelagic zone of oceans, seas, or bodies of fresh water. It is a description of life-style rather than a genetic classification. They are widely considered to be some of the most important organisms on Earth, due to the food supply they provide to most aquatic life.
The name plankton is derived from the Greek word πλανκτος ("planktos"), meaning "wanderer" or "drifter".While some forms of plankton are capable of independent movement and can swim up to several hundreds of meters vertically in a single day (a behavior called diel vertical migration), their horizontal position is primarily determined by currents in the body of water they inhabit. By definition, organisms classified as plankton are unable to resist ocean currents. This is in contrast to nekton organisms that can swim against the ambient flow of the water environment and control their position (e.g. squid, fish, and marine mammals).
Within the plankton, itself, holoplankton are those organisms that spend their entire life cycle as part of the plankton (e.g. most algae, copepods, salps, and some jellyfish). By contrast, meroplankton are those organisms that are only planktonic for part of their lives (usually the larval stage), and then graduate to either the nekton or a benthic (sea floor) existence. Examples of meroplankton include the larvae of sea urchins, starfish, crustaceans, marine worms, and most fish.
Plankton abundance and distribution are strongly dependent on factors such as ambient nutrients concentrations, the physical state of the water column, and the abundance of other plankton.
The study of plankton is termed planktology. Individual plankton are referred to as plankters.
Quorum sensing is the process by which many bacteria coordinate their gene expression according to the local density of their population by producing signaling molecules.
Quorum sensing coordinates certain behavior or actions between bacteria, based on the local density of the bacterial population. Quorum sensing can occur within a single bacterial species as well as between diverse species, and can regulate a host of different processes, essentially serving as a simple communication network.
For example, opportunistic bacteria, such as Pseudomonas aeruginosa can grow within a host without harming it, until they reach a certain concentration. Then they become aggressive, their numbers sufficient to overcome the host's immune system and form a biofilm, leading to disease. It is hoped that the therapeutic enzymatic degradation of the signalling molecules will prevent the formation of such biofilms and possibly weaken established biofilms. Disrupting the signalling process in this way is called quorum quenching.
Bacteria that use quorum sensing produce and secrete certain signaling compounds (called autoinducers or pheromones), one example of which are N-acyl homoserine lactones (AHL). These bacteria also have a receptor that can specifically detect the AHL (inducer). When the inducer binds the receptor, it activates transcription of certain genes, including those for inducer synthesis. There is a low likelihood of a bacterium detecting its own secreted AHL.
When only a few other bacteria of the same kind are in the vicinity, diffusion reduces the concentration of the inducer in the surrounding medium to almost zero, so the bacteria produce little inducer. With many bacteria of the same kind, the concentration of the inducer passes a threshold, whereupon more inducer is synthesised. This forms a positive feedback loop, and the receptor becomes fully activated. This induces the up regulation of other specific genes, such as luciferase in V. fischeri. This is useful since a single V. fischeri bacterium that is luminescent would have no evolutionary advantage and would be wasting energy.
In Escherichia coli, AI-2 is produced and processed by the lsr operon. Part of it encodes an ABC transporter which imports AI-2 into the cells during the early stationary (latent) phase of growth. AI-2 is then phosphorylated by the LsrK kinase, and the newly produced phospho-AI-2 can either be internalized or used to suppress LsrR, a repressor of the lsr operon (thereby activating the operon). Transcription of the lsr operon is also thought to be inhibited by dihydroxyacetone phosphate (DHAP) through its competitive binding to LsrR. Glyceraldehyde 3-phosphate has also been shown to inhibit the lsr operon through cAMP-CAPK-mediated inhibition. This explains why when grown with glucose E. coli will lose the ability to internalize AI-2 (because of catabolite repression). When grown normally, AI-2 presence is transient.
Three-dimensional structures of proteins involved in quorum-sensing were first published in 2001, when the crystal structures of three LuxS orthologs were determined by X-ray crystallography. In 2002, the crystal structure of the receptor LuxP of Vibrio harveyi with its inducer AI-2 (which is one of the few biomolecules containing boron) bound to it was also determined. AI-2 signalling is conserved among many bacterial species, including E. coli, an enteric bacterium and model organism for Gram-negative bacteria. Although this conservation has suggested that autoinducer-2 could be used for widespread interspecies communication, a comparative genomic and phylogenetic analysis of 138 genomes of bacteria, archaea, and eukaryotes did not support the concept of a multispecies signaling system relying on AI-2 outside Vibrio species.
Quorum sensing is the process by which many bacteria coordinate their gene expression according to the local density of their population by producing signaling molecules.
Consequences
Quorum sensing coordinates certain behavior or actions between bacteria, based on the local density of the bacterial population. Quorum sensing can occur within a single bacterial species as well as between diverse species, and can regulate a host of different processes, essentially serving as a simple communication network.
For example, opportunistic bacteria, such as Pseudomonas aeruginosa can grow within a host without harming it, until they reach a certain concentration. Then they become aggressive, their numbers sufficient to overcome the host's immune system and form a biofilm, leading to disease. It is hoped that the therapeutic enzymatic degradation of the signalling molecules will prevent the formation of such biofilms and possibly weaken established biofilms. Disrupting the signalling process in this way is called quorum quenching.
A phenotype is any observed quality of an organism, such as its morphology, development, or behavior, as opposed to its genotype - the inherited instructions it carries, which may or may not be expressed. This genotype-phenotype distinction was proposed by Wilhelm Johannsen in 1911 to make clear the difference between an organism's heredity and what that heredity produces. The distinction is similar to that proposed by August Weismann, who distinguished between germ plasm (heredity) and somatic cells (the body). A more modern version is Francis Crick's Central dogma of molecular biology.
Despite its seemingly straightforward definition, the concept of the phenotype has some hidden subtleties. First, most of the molecules and structures coded by the genetic material are not visible in the appearance of an organism, yet are part of the phenotype. Human blood groups are an example. So, by extension, the term phenotype must include characteristics that can be made visible by some technical procedure. A further, and more radical, extension would add inherited behaviour to the phenotype.
A cell membrane defines a boundary between the living cell and its environment. It consists of lipids, proteins,carbohydrates etc. Lipids and proteins are dominant components of membranes. One of the principal types of lipids in membranes is phospholipid. A phospholipid molecule has a polar hydrophilic head group and two hydrophobic hydrocarbon tails. When a quantity of lipid molecules disperse in water, they will assemble themselves into a bilayer in which the hydrophilic heads shield the hydrophobic tails from the water surroundings because of the hydrophobic forces.
The most widely accepted model for cell membranes is the fluid mosaic model proposed by Singer and Nicolson in 1972 [Science 175 (1972) 720]. In this model, the cell membrane is considered as a lipid bilayer where the lipid molecules can move freely in the membrane surface like fluid, while the proteins are embedded in the lipid bilayer. Some proteins are called integral membrane proteins because they traverse entirely in the lipid bilayer and play the role of information and matter communications between the interior of the cell and its outer environment. The others are called peripheral membrane proteins because they are partially embedded in the bilayer and accomplish the other biological functions. Beneath the lipid membrane, the membrane skeleton, a network of proteins, links with the proteins in the lipid membrane. A more recent model of the cell membrane as a self-organising, dynamic structure in which phospholipids flow in geometric patterns identical to those associated with 'Benard' convection' imbue the cell with it's elastic properties is gaining wide acceptance.
Phospholipids are a class of lipids, and a major component of all biological membranes, along with glycolipids, cholesterol and proteins. Understanding of the aggregation properties of these molecules is known as lipid polymorphism and forms part of current academic research.
A fluid is defined as a substance that continually deforms (flows) under an applied shear stress regardless of how small the applied stress. All liquids and all gases are fluids. Fluids are a subset of the phases of matter and include liquids, gases, plasmas and, to some extent, plastic solids. The term "fluid" is often used as being synonymous with "liquid". This can be erroneous and sometimes clearly inappropriate - such as when referring to a liquid which does not or should not involve the gaseous state. "Brake fluid" is hydraulic oil which will not perform its required function if gas is present. The medical profession relies on the term "fluids" in dietary references ("take plenty of fluids") where the presence of gases is irrelevant or even possibly dangerous.
Liquids form a free surface (that is, a surface not created by the container) while gases do not. The distinction between solids and fluid is not entirely obvious. The distinction is made by evaluating the viscosity of the substance. Silly Putty can be considered either a solid or a fluid, depending on the time period over which it is observed.
Fluids display such properties as:
not resisting deformation, or resisting it only lightly (viscosity), and
the ability to flow (also described as the ability to take on the shape of the container).
An Integral Membrane Protein (IMP) is a protein molecule (or assembly of proteins) that is permanently attached to the biological membrane. Such proteins can be separated from the biological membranes only using detergents, nonpolar solvents, or sometimes denaturing agents.
IMPs comprise a very significant fraction of the proteins encoded in the genome.
Structure
Three-dimensional structures of only ~160 different integral membrane proteins are currently determined at atomic resolution by X-ray crystallography or Nuclear magnetic resonance spectroscopy due to the difficulties with extraction and crystallization. In addition, structures of many water-soluble domains of IMPs are available in the Protein Data Bank. Their membrane-anchoring α-helices have been removed to facilitate the extraction and crystallization.
IMPs can be divided into two groups:
Transmembrane proteins
Integral monotopic proteins
Peripheral membrane proteins are proteins that adhere only temporarily to the biological membrane with which they are associated. These molecules attach to integral membrane proteins, or penetrate the peripheral regions of the lipid bilayer. The regulatory protein subunits of many ion channels and transmembrane receptors, for example, may be defined as peripheral membrane proteins. In contrast to integral membrane proteins, peripheral membrane proteins tend to collect in the water-soluble component, or fraction, of all the proteins extracted during a protein purification procedure. Proteins with GPI anchors are an exception to this rule and can have purification properties similar to those of integral membrane proteins.
The reversible attachment of proteins to biological membranes has shown to regulate cell signaling and many other important cellular events, through a variety of mechanisms. For example, the close association between many enzymes and biological membranes may bring them into close proximity with their lipid substrate(s).Membrane binding may also promote rearrangement, dissociation, or conformational changes within many protein structural domains, resulting in an activation of their biological activity.Additionally, the positioning of many proteins are localized to either the inner or outer surfaces or leaflets of their resident membrane. This facilitates the assembly of multi-protein complexes by increasing the probability of any appropriate protein-protein interactions.
A membrane protein is a protein molecule that is attached to, or associated with the membrane of a cell or an organelle. More than half of all proteins interact with membranes. Membrane proteins can be classified into two groups, based on the strength of their association with the membrane.
Classification of membrane proteins to integral and peripheral does not include some polypeptide toxins, such as colicin A or alpha-hemolysin, and certain proteins involved in apoptosis. These proteins are water-soluble but can aggregate and associate irreversibly with the lipid bilayer and form alpha-helical or beta-barrel transmembrane channels. An alternative classification is to divide all membrane proteins to integral and amphitropic. The amphitropic are proteins that can exist in two alternative states: a water-soluble and a lipid bilayer-bound, whereas integral proteins can be found only in the membrane-bound state. The amphitropic protein category includes water-soluble channel-forming polypeptide toxins, which associate irreversibly with membranes, but excludes peripheral proteins that interact with other membrane proteins rather than with lipid bilayer.
