What is Endocytosis

Endocytosis is a process whereby cells absorb material (molecules such as proteins) from the outside by engulfing it with their cell membrane. It is used by all cells of the body because most substances important to them are large polar molecules, and thus cannot pass through the hydrophobic plasma membrane. The function of endocytosis is the opposite of exocytosis.

Types

The absorption of material from the outside environment of the cell is commonly divided into two processes: phagocytosis and pinocytosis.

Phagocytosis (literally, cell-eating) is the process by which cells ingest large objects, such as cells which have undergone apoptosis, bacteria, or viruses. The membrane folds around the object, and the object is sealed off into a large vacuole known as a phagosome.

Pinocytosis (literally, cell-drinking) is a synonym for endocytosis. This process is concerned with the uptake of solutes and single molecules such as proteins.

Receptor-mediated endocytosis is a more specific active event where the cytoplasm membrane folds inward to form coated pits. These inward budding vesicles bud to form cytoplasmic vesicles.

Endocytosis pathways

There are three types of endocytosis: namely, macropinocytosis, clathrin-mediated endocytosis, and caveolar endocytosis.

Macropinocytosis is the invagination of the cell membrane to form a pocket which then pinches off into the cell to form a vesicle filled with extracellular fluid (and molecules within it). The filling of the pocket occurs in a non-specific manner. The vesicle then travels into the cytosol and fuses with other vesicles such as endosomes and lysosomes.

Clathrin-mediated endocytosis is the specific uptake of large extracellular molecules such as proteins, membrane localized receptors and ion-channels. These receptors are associated with the cytosolic protein clathrin which initiates the formation of a vesicle by forming a crystalline coat on the inner surface of the cell's membrane.

Caveolae consist of the protein caveolin-1 with a bilayer enriched in cholesterol and glycolipids. Caveolae are flask shaped pits in the membrane that resemble the shape of a cave (hence the name caveolae). Uptake of extracellular molecules are also believed to be specifically mediated via receptors in caveolae.

Clathrin-mediated endocytosis

The major route for endocytosis in most cells, and the best understood, is that mediated by the molecule clathrin. This large protein assists in the formation of a coated pit on the inner surface of the plasma membrane of the cell. This pit then buds into the cell to form a coated vesicle in the cytoplasm of the cell. In so doing, it brings into the cell not only a small area of the surface of the cell but also a small volume of fluid from outside the cell.

Vesicles selectively concentrate and exclude certain proteins during formation and are not representative of the membrane as a whole. AP2 adaptors are multisubunit complexes that performs this function at the plasma membrane. The best understood receptors which are found concentrated in coated vesicles of mammalian cells are the LDL receptor (which removes LDL from the blood circulation), the transferrin receptor (which brings ferric ions bound by transferrin into the cell) and certain hormone receptors (such as that for EGF).

At any one moment, about 2% of the plasma membrane of a fibroblast is made up of coated pits. As a coated pit has a life of about a minute before it buds into the cell, a fibroblast takes up its surface by this route about once every 50 minutes. Coated vesicles formed from the plasma membrane have a diameter of about 100nm and a life time measured in a few seconds. Once the coat has been shed, the remaining vesicle fuses with endosomes and proceeds down the endocytic pathway. The actual budding-in process, whereby a pit is converted to a vesicle, is carried out by clathrin assisted by a set of cytoplasmic proteins which includes dynamin and adaptors such as adaptin.

Coated pits and vesicles were first seen in thin sections of tissue in the electron microscope by Thomas Roth and Keith Porter in 1964. The importance of them for the clearance of LDL from blood was discovered by R. G Anderson, Michael S. Brown and Joseph L. Goldstein in 1976. Coated vesicles were first purified by Barbara Pearse, who discovered the clathrin coat molecule, also in 1976.

H5N1 VIRUS

Influenza A virus subtype H5N1, also known as A(H5N1) or simply H5N1, is a subtype of the Influenza A virus which can cause illness in humans and many other animal species. A bird-adapted strain of H5N1, called HPAI A(H5N1) for "highly pathogenic avian influenza virus of type A of subtype H5N1", is the causative agent of H5N1 flu, commonly known as "avian influenza" or "bird flu". It is endemic in many bird populations, especially in Southeast Asia. One strain of HPAI A(H5N1) is spreading globally after first appearing in Asia. It is epizootic (an epidemic in nonhumans) and panzootic (affecting animals of many species, especially over a wide area), killing tens of millions of birds and spurring the culling of hundreds of millions of others to stem its spread. Most mentions of "bird flu" and H5N1 in the media refer to this strain.

HPAI A(H5N1) is an avian disease. There is no evidence of efficient human-to-human transmission or of airborne transmission of HPAI A(H5N1) to humans. In almost all cases, those infected with H5N1 had extensive physical contact with infected birds. Still, around 60% of humans known to have been infected with the current Asian strain of HPAI A(H5N1) have died from it, and H5N1 may mutate or reassort into a strain capable of efficient human-to-human transmission. In 2003, world-renowned virologist Robert Webster published an article titled "The world is teetering on the edge of a pandemic that could kill a large fraction of the human population" in American Scientist. He called for adequate resources to fight what he sees as a major world threat to possibly billions of lives. On September 29, 2005, David Nabarro, the newly-appointed Senior United Nations System Coordinator for Avian and Human Influenza, warned the world that an outbreak of avian influenza could kill anywhere between 5 million and 150 million people. Experts have identified key events (creating new clades, infecting new species, spreading to new areas) marking the progression of an avian flu virus towards becoming pandemic, and many of those key events have occurred more rapidly than expected.

Due to the high lethality and virulence of HPAI A(H5N1), its endemic presence, its increasingly large host reservoir, and its significant ongoing mutations, the H5N1 virus is the world's largest current pandemic threat, and billions of dollars are being spent researching H5N1 and preparing for a potential influenza pandemic. At least 12 companies and 17 governments are developing pre-pandemic influenza vaccines in 28 different clinical trials that, if successful, could turn a deadly pandemic infection into a nondeadly one. Full-scale production of a vaccine that could prevent any illness at all from the strain would require at least three months after the virus's emergence to begin, but it is hoped that vaccine production could increase until one billion doses were produced by one year after the initial identification of the virus.

H5N1 may cause more than one influenza pandemic as it is expected to continue mutating in birds regardless of whether humans develop herd immunity to a future pandemic strain. Influenza pandemics from its genetic offspring may include influenza A virus subtypes other than H5N1. While genetic analysis of the H5N1 virus shows that influenza pandemics from its genetic offspring can easily be far more lethal than the Spanish Flu pandemic, planning for a future influenza pandemic is based on what can be done and there is no higher Pandemic Severity Index level than a Category 5 pandemic which, roughly speaking, is any pandemic as bad as the Spanish flu or worse; and for which all intervention measures are to be used.

Genetics

The first known strain of HPAI A(H5N1) (called A/chicken/Scotland/59) killed two flocks of chickens in Scotland in 1959; but that strain was very different from the current highly pathogenic strain of H5N1. The dominant strain of HPAI A(H5N1) in 2004 evolved from 1999 to 2002 creating the Z genotype. It has also been called "Asian lineage HPAI A(H5N1)".

Asian lineage HPAI A(H5N1) is divided into two antigenic clades. "Clade 1 includes human and bird isolates from Vietnam, Thailand, and Cambodia and bird isolates from Laos and Malaysia. Clade 2 viruses were first identified in bird isolates from China, Indonesia, Japan, and South Korea before spreading westward to the Middle East, Europe, and Africa. The clade 2 viruses have been primarily responsible for human H5N1 infections that have occurred during late 2005 and 2006, according to WHO. Genetic analysis has identified six subclades of clade 2, three of which have a distinct geographic distribution and have been implicated in human infections: Map

• Subclade 1, Indonesia
  
• Subclade 2, Europe, Middle East, and Africa (called EMA)
  
• Subclade 3, China".
  
• 
  

A 2007 study focused on the EMA subclade has shed further light on the EMA mutations. "The 36 new isolates reported here greatly expand the amount of whole-genome sequence data available from recent avian influenza (H5N1) isolates. Before our project, GenBank contained only 5 other complete genomes from Europe for the 2004–2006 period, and it contained no whole genomes from the Middle East or northern Africa. Our analysis showed several new findings. First, all European, Middle Eastern, and African samples fall into a clade that is distinct from other contemporary Asian clades, all of which share common ancestry with the original 1997 Hong Kong strain. Phylogenetic trees built on each of the 8 segments show a consistent picture of 3 lineages, as illustrated by the HA tree shown in Figure 1. Two of the clades contain exclusively Vietnamese isolates; the smaller of these, with 5 isolates, we label V1; the larger clade, with 9 isolates, is V2. The remaining 22 isolates all fall into a third, clearly distinct clade, labeled EMA, which comprises samples from Europe, the Middle East, and Africa. Trees for the other 7 segments display a similar topology, with clades V1, V2, and EMA clearly separated in each case. Analyses of all available complete influenza (H5N1) genomes and of 589 HA sequences placed the EMA clade as distinct from the major clades circulating in People's Republic of China, Indonesia, and Southeast Asia."

Terminology

H5N1 isolates are identified like this actual HPAI A(H5N1) example, A/chicken/Nakorn-Patom/Thailand/CU-K2/04(H5N1):

• A stands for the species of influenza (A, B or C).
  
• chicken is the species the isolate was found in
  
• Nakorn-Patom/Thailand is the place this specific virus was isolated
  
• CU-K2 identifies it from other influenza viruses isolated at the same place
  
• 04 represents the year 2004
  
• H5 stands for the fifth of several known types of the protein hemagglutinin.
  
• N1 stands for the first of several known types of the protein neuraminidase.
  
• 
  

As with other avian flu viruses, H5N1 has strains called "highly pathogenic" (HP) and "low-pathogenic" (LP). Avian influenza viruses that cause HPAI are highly virulent, and mortality rates in infected flocks often approach 100%. LPAI viruses have negligible virulence, but these viruses can serve as progenitors to HPAI viruses. The current strain of H5N1 responsible for the deaths of birds across the world is an HPAI strain; all other current strains of H5N1, including a North American strain that causes no disease at all in any species, are LPAI strains. All HPAI strains identified to date have involved H5 and H7 subtypes. The distinction concerns pathogenicity in poultry, not humans. Normally a highly pathogenic avian virus is not highly pathogenic to either humans or non-poultry birds. This current deadly strain of H5N1 is unusual in being deadly to so many species, including some, like domestic cats, never previously susceptible to any influenza virus.

Properties of H5N1

H5N1 is easily transmissible between birds facilitating a potential global spread of H5N1. While H5N1 undergoes mutation and reassortment, creating variations which can infect species not previously known to carry the virus, not all of these variant forms can infect humans. H5N1 as an avian virus preferentially binds to a type of galactose receptors that populate the avian respiratory tract from the nose to the lungs and are virtually absent in humans, occurring only in and around the alveoli, structures deep in the lungs where oxygen is passed to the blood. Therefore, the virus is not easily expelled by coughing and sneezing, the usual route of transmission.

H5N1 is mainly spread by domestic poultry, both through the movements of infected birds and poultry products and through the use of infected poultry manure as fertilizer or feed. Humans with H5N1 have typically caught it from chickens, which were in turn infected by other poultry or waterfowl. Migrating waterfowl (wild ducks, geese and swans) carry H5N1, often without becoming sick. Many species of birds and mammals can be infected with HPAI A(H5N1), but the role of animals other than poultry and waterfowl as disease-spreading hosts is unknown.

Virulence

H5N1 has mutated into a variety of strains with differing pathogenic profiles, some pathogenic to one species but not others, some pathogenic to multiple species. Each specific known genetic variation is traceable to a virus isolate of a specific case of infection. Through antigenic drift, H5N1 has mutated into dozens of highly pathogenic varieties divided into genetic clades which are known from specific isolates, but all currently belonging to genotype Z of avian influenza virus H5N1, now the dominant genotype. H5N1 isolates found in Hong Kong in 1997 and 2001 were not consistently transmitted efficiently among birds and did not cause significant disease in these animals. In 2002 new isolates of H5N1 were appearing within the bird population of Hong Kong. These new isolates caused acute disease, including severe neurological dysfunction and death in ducks. This was the first reported case of lethal influenza virus infection in wild aquatic birds since 1961. Genotype Z emerged in 2002 through reassortment from earlier highly pathogenic genotypes of H5N1 that first infected birds in China in 1996, and first infected humans in Hong Kong in 1997.Genotype Z is endemic in birds in Southeast Asia, has created at least two clades that can infect humans, and is spreading across the globe in bird populations. Mutations are occurring within this genotype that are increasing their pathogenicity. Birds are also able to shed the virus for longer periods of time before their death, increasing the transmissibility of the virus.

Transmission and host range

Infected birds transmit H5N1 through their saliva, nasal secretions, feces and blood. Other animals may become infected with the virus through direct contact with these bodily fluids or through contact with surfaces contaminated with them. H5N1 remains infectious after over 30 days at 0 °C ( 32.0 °F) (over one month at freezing temperature) or 6 days at 37 °C ( 98.6 °F) (one week at human body temperature) so at ordinary temperatures it lasts in the environment for weeks. In Arctic temperatures, it doesn't degrade at all.

Because migratory birds are among the carriers of the highly pathogenic H5N1 virus, it is spreading to all parts of the world. H5N1 is different from all previously known highly pathogenic avian flu viruses in its ability to be spread by animals other than poultry.

In October 2004, researchers discovered that H5N1 is far more dangerous than was previously believed. Waterfowl were revealed to be directly spreading the highly pathogenic strain of H5N1 to chickens, crows, pigeons, and other birds, and the virus was increasing its ability to infect mammals as well. From this point on, avian flu experts increasingly referred to containment as a strategy that can delay, but not ultimately prevent, a future avian flu pandemic.

"Since 1997, studies of influenza A (H5N1) indicate that these viruses continue to evolve, with changes in antigenicity and internal gene constellations; an expanded host range in avian species and the ability to infect felids; enhanced pathogenicity in experimentally infected mice and ferrets, in which they cause systemic infections; and increased environmental stability."

The New York Times, in an article on transmission of H5N1 through smuggled birds, reports Wade Hagemeijer of Wetlands International stating, "We believe it is spread by both bird migration and trade, but that trade, particularly illegal trade, is more important"

Influenza viruses have a relatively high mutation rate that is characteristic of RNA viruses. The segmentation of its genome facilitates genetic recombination by segment reassortment in hosts infected with two different influenza viruses at the same time. A previously uncontagious strain may then be able to pass between humans, one of several possible paths to a pandemic.

The ability of various influenza strains to show species-selectivity is largely due to variation in the hemagglutinin genes. Genetic mutations in the hemagglutinin gene that cause single amino acid substitutions can significantly alter the ability of viral hemagglutinin proteins to bind to receptors on the surface of host cells. Such mutations in avian H5N1 viruses can change virus strains from being inefficient at infecting human cells to being as efficient in causing human infections as more common human influenza virus types. This doesn't mean that one amino acid substitution can cause a pandemic, but it does mean that one amino acid substitution can cause an avian flu virus that is not pathogenic in humans to become pathogenic in humans.

H3N2 ("swine flu") is endemic in pigs in China, and has been detected in pigs in Vietnam, increasing fears of the emergence of new variant strains. The dominant strain of annual flu virus in January 2006 was H3N2, which is now resistant to the standard antiviral drugs amantadine and rimantadine. The possibility of H5N1 and H3N2 exchanging genes through reassortment is a major concern. If a reassortment in H5N1 occurs, it might remain an H5N1 subtype, or it could shift subtypes, as H2N2 did when it evolved into the Hong Kong Flu strain of H3N2.

Both the H2N2 and H3N2 pandemic strains contained avian influenza virus RNA segments. "While the pandemic human influenza viruses of 1957 (H2N2) and 1968 (H3N2) clearly arose through reassortment between human and avian viruses, the influenza virus causing the 'Spanish flu' in 1918 appears to be entirely derived from an avian source".

Humans and H5N1





The earliest infections of humans by H5N1 coincided with an epizootic (an epidemic in nonhumans) of H5N1 influenza in Hong Kong's poultry population. This panzootic (a disease affecting animals of many species, especially over a wide area) outbreak was stopped by the killing of the entire domestic poultry population within the territory.

Symptoms in humans

Avian influenza hemagglutinin bind alpha 2-3 sialic acid receptors while human influenza hemagglutinin bind alpha 2-6 sialic acid receptors. Usually other differences also exist. There is as yet no human form of H5N1, so all humans who have caught it so far have caught avian H5N1.

In general, humans who catch a humanized Influenza A virus (a human flu virus of type A) usually have symptoms that include fever, cough, sore throat, muscle aches, conjunctivitis and, in severe cases, breathing problems and pneumonia that may be fatal. The severity of the infection depends to a large part on the state of the infected person's immune system and whether the victim has been exposed to the strain before (in which case they would be partially immune). No one knows if these or other symptoms will be the symptoms of a humanized H5N1 flu.

The mortality rate of highly pathogenic H5N1 avian influenza in a human is high; WHO data indicates that 60% of cases classified as H5N1 resulted in death. In one case, a boy with H5N1 experienced diarrhea followed rapidly by a coma without developing respiratory or flu-like symptoms. There have been studies of the levels of cytokines in humans infected by the H5N1 flu virus. Of particular concern is elevated levels of tumor necrosis factor-alpha, a protein that is associated with tissue destruction at sites of infection and increased production of other cytokines. Flu virus-induced increases in the level of cytokines is also associated with flu symptoms including fever, chills, vomiting and headache. Tissue damage associated with pathogenic flu virus infection can ultimately result in death. The inflammatory cascade triggered by H5N1 has been called a 'cytokine storm' by some, because of what seems to be a positive feedback process of damage to the body resulting from immune system stimulation. H5N1 induces higher levels of cytokines than the more common flu virus types

Protecting virus for flu

Nerve Impulse

The conduction of nerve impulses relies upon the movement of electrically charge ions across the nerve cell membrane. When a nerve is resting, or polorized, there are more potassium ions than sodium ions inside the cell, with an opposite ratio outside. Sodium ions are actively kept out of the cell by an energy consuming pump mechanism. This maintains a negative charge on the inside of the cell and a positive charge on the outside. When an impulse travels along the nerve, sodium ions flood into the cell and make the inside of the cell positive with respect to the outside. This produces a rise in the electrical potential across the cell membrane. After the impulse has passed, potassium ions leave the cell, restoring the negative charge within the cell and the positive charge outside it. While this resting situation is being restored another impulse cannot be generated.

Ion Conductances

The generation of action potentials is mainly due to the changes of sodium (Na+) and potassium (K+) conductances. Figure 2 shows the concentrations of Na+ and K+ ions on both sides of a nerve membrane. For Na+, its concentration on the extracellular side is much higher than inside. We immediately notice that Na+ ions are far from electrochemical equilibrium -- both the electric force due to electric potential difference and the chemical force due to ion concentration difference are pointing inward. How could the nerve membrane maintain such a stable state? This is because the conductance of Na+ ions in the membrane is very small at the resting membrane potential. Although the inward driving force is large, the resulting Na+ influx is small. This small influx can be balanced by a slow ion transport process, the Na+-K+ pump, which moves the Na+ ions outward and simultaneously the K+ ions inward.

The conductance of Na+ ions may change dramatically with the membrane potential as demonstrated by voltage clamp experiments, in which the membrane potential is displaced to a new value and maintained there . Because ions carry charges, the movement of ions across the membrane will change the membrane potential. To maintain a constant membrane potential, the voltage clamp circuit must generate electric currents to neutralize the membrane potential change caused by the ionic flux. Thus, the ion current through the membrane is reflected in the electric current of the voltage clamp circuit outside the membrane

MALARIAL INFECTION ANIMATION

Malaria is a vector-borne infectious disease caused by protozoan parasites. It is widespread in tropical and subtropical regions, including parts of the Americas, Asia, and Africa. Each year, it causes disease in approximately 650 million people and kills between one and three million, most of them young children in Sub-Saharan Africa. Malaria is commonly-associated with poverty, but is also a cause of poverty and a major hindrance to economic development.

Malaria is one of the most common infectious diseases and an enormous public-health problem. The disease is caused by protozoan parasites of the genus Plasmodium. The most serious forms of the disease are caused by Plasmodium falciparum and Plasmodium vivax, but other related species (Plasmodium ovale, Plasmodium malariae, and sometimes Plasmodium knowlesi) can also infect humans. This group of human-pathogenic Plasmodium species is usually referred to as malaria parasites.

Malaria parasites are transmitted by female Anopheles mosquitoes. The parasites multiply within red blood cells, causing symptoms that include symptoms of anemia (light headedness, shortness of breath, tachycardia etc.), as well as other general symptoms such as fever, chills, nausea, flu-like illness, and in severe cases, coma and death. Malaria transmission can be reduced by preventing mosquito bites with mosquito nets and insect repellents, or by mosquito control by spraying insecticides inside houses and draining standing water where mosquitoes lay their eggs.

Pathogenesis

Malaria in humans develops via two phases: an exoerythrocytic (hepatic) and an erythrocytic phase. When an infected mosquito pierces a person's skin to take a blood meal, sporozoites in the mosquito's saliva enter the bloodstream and migrate to the liver. Within 30 minutes of being introduced into the human host, they infect hepatocytes, multiplying asexually and asymptomatically for a period of 6–15 days. During this so-called dormant time in the liver, the sporozoites are often referred to as hypnozoites. Once in the liver these organisms differentiate to yield thousands of merozoites which, following rupture of their host cells, escape into the blood and infect red blood cells, thus beginning the erythrocytic stage of the life cycle.The parasite escapes from the liver undetected by wrapping itself in the cell membrane of the infected host liver cell.

Within the red blood cells the parasites multiply further, again asexually, periodically breaking out of their hosts to invade fresh red blood cells. Several such amplification cycles occur. Thus, classical descriptions of waves of fever arise from simultaneous waves of merozoites escaping and infecting red blood cells.

Some P. vivax and P. ovale sporozoites do not immediately develop into exoerythrocytic-phase merozoites, but instead produce hypnozoites that remain dormant for periods ranging from several months (6–12 months is typical) to as long as three years. After a period of dormancy, they reactivate and produce merozoites. Hypnozoites are responsible for long incubation and late relapses in these two species of malaria.

The parasite is relatively protected from attack by the body's immune system because for most of its human life cycle it resides within the liver and blood cells and is relatively invisible to immune surveillance. However, circulating infected blood cells are destroyed in the spleen. To avoid this fate, the P. falciparum parasite displays adhesive proteins on the surface of the infected blood cells, causing the blood cells to stick to the walls of small blood vessels, thereby sequestering the parasite from passage through the general circulation and the spleen. This "stickiness" is the main factor giving rise to hemorrhagic complications of malaria. High endothelial venules (the smallest branches of the circulatory system) can be blocked by the attachment of masses of these infected red blood cells. The blockage of these vessels causes symptoms such as in placental and cerebral malaria. In cerebral malaria the sequestrated red blood cells can breach the blood brain barrier possibly leading to coma.

Although the red blood cell surface adhesive proteins (called PfEMP1, for Plasmodium falciparum erythrocyte membrane protein 1) are exposed to the immune system they do not serve as good immune targets because of their extreme diversity; there are at least 60 variations of the protein within a single parasite and perhaps limitless versions within parasite populations. Like a thief changing disguises or a spy with multiple passports, the parasite switches between a broad repertoire of PfEMP1 surface proteins, thus staying one step ahead of the pursuing immune system.

Some merozoites turn into male and female gametocytes. If a mosquito pierces the skin of an infected person, it potentially picks up gametocytes within the blood. Fertilization and sexual recombination of the parasite occurs in the mosquito's gut, thereby defining the mosquito as the definitive host of the disease. New sporozoites develop and travel to the mosquito's salivary gland, completing the cycle. Pregnant women are especially attractive to the mosquitoes, and malaria in pregnant women is an important cause of stillbirths, infant mortality and low birth weight

Four-Gene DNA Vaccine

Recombinant DNA Technology

Recombinant DNA is a form of artificial DNA which is engineered through the combination or insertion of one or more DNA strands, thereby combining DNA sequences which would not normally occur together. In terms of genetic modification, recombinant DNA is produced through the addition of relevant DNA into an existing organismal genome, such as the plasmid of bacteria, to code for or alter different traits for a specific purpose, such as immunity. It differs from genetic recombination, in that it does not occur through processes within the cell or ribosome, but is exclusively engineered.

The Recombinant DNA technique was engineered by Stanley Norman Cohen and Herbert Boyer in 1973. They published their findings in a 1974 paper entitled "Construction of Biologically Functional Bacterial Plasmids in vitro", which described a technique to isolate and amplify genes or DNA segments and insert them into another cell with precision, creating a transgenic bacterium. Recombinant DNA technology was made possible by the discovery of restriction endonucleases by Werner Arber, Daniel Nathans, and Hamilton Smith, for which they received the 1978 Nobel Prize in Medicine.

Introduction

Because of the importance in DNA in the replication of new structures and characteristics of living organisms, it has widespread importance in recapitulating via viral or non-viral vectors, both desirable and undesirable characteristics of a species to achieve characteristic change or to counteract effects caused by genetic or imposed disorders that have effects upon cellular or organismal processes. Through the use of recombinant DNA, genes that are identified as important can be amplified and isolated for use in other species or applications, where there may be some form of genetic illness or discrepancy, and provides a different approach to complex biological problem solving.

Applications and methods
Cloning and relation to plasmids

The use of cloning is interrelated with Recombinant DNA in classical biology, as the term "clone" refers to a cell or organism derived from a parental organism, with modern biology referring to the term as a collection of cells derived from the same cell which remain identical. In the classical instance, the use of recombinant DNA provides the initial cell from which the host organism is then expected to recapitulate when it undergoes further cell division, with bacteria remaining a prime example due to the use of viral vectors in medicine which contain recombinant DNA inserted into a structure known as a plasmid.

Plasmids are extrachromosomal self replicating circular forms of DNA present in most bacteria, such as Escherichia coli (E. Coli), contain genes related to catabolism and metabolic activity, and allow the carrier bacterium to survive and reproduce in conditions present within other species and environments. These genes represent characteristics of resistance to bacteriophages and antibiotics and some heavy metals, but can also be fairly easily removed or separated from the plasmid by restriction endonucleases, which regularly produce "sticky ends" and allow the attachment of a selected segment of DNA, which codes for more "reparative" substances, such as peptide hormone medications including insulin, growth hormone, and oxytocin. In the introduction of useful genes into the plasmid, the bacteria is then used as a viral vector, which is encouraged to reproduce so as to recapitulate the altered DNA within other cells it infects and increase the amount of cells with the recombinant DNA present within them.

The use of plasmids is also key within gene therapy, where their related viruses are used as cloning vectors or carriers, which are means of transporting and passing on genes in recombinant DNA through viral reproduction throughout an organism. As a general definition of plasmids, the definition is that they contain three common features -- a replicator, selectable marker and a cloning site.The replicator or "ori" refers to the origin of replication with regards to location and bacteria where replication begins. The marker refers to a gene which usually contains resistance to an antibiotic, but may also refer to a gene which is attached alongside the desired one, such as that which confers luminescence to allow identification of successfully recombined DNA. The cloning site is a sequence of nucleotides representing one or more positions where cleavage by restriction endonucleases occurs. Most eukaryotes do not maintain canonical plasmids; yeast is a notable exception. In addition, the Ti plasmid of the bacterium Agrobacterium tumefaciens can be used to integrate foreign DNA into the genomes of many plants. Other methods of introducing or creating recombinant DNA in eukaryotes include homologous recombination and transfection with modified viruses.

Chimeric plasmids

When recombinant DNA is then further altered or changed to host additional strands of DNA, the molecule formed is referred to as "chimeric" DNA molecule, with reference to the mythological chimera which consisted as a composite of several animals. The presence of chimeric plasmid molecules is somewhat regular in occurrence as throughout the lifetime of an organism the propagation by vectors ensures the presence of hundreds of thousands of organismal and bacterial cells which all contain copies of the original chimeric DNA.

In the production of chimeric plasmids, the processes involved can be somewhat uncertainas the intended outcome of the addition of foreign DNA may not always be achieved and may result in the formation of unusable plasmids. Initially, the plasmid structure is linearised to allow the addition by bonding of complimentary foreign DNA strands to single-stranded "overhangs" or "sticky ends" present at the ends of the DNA molecule from staggered, or "S shaped" cleavages produced by restriction endonucleases.

A common vector used for the donation of plasmids originally was the bacterium Escherichia coli and later, the EcoRI derivative which was used for it's versatility with addition of new DNA by "relaxed" replication when inhibited by chloramphenicol and spectinomycin; later being replaced by the pBR322 plasmid.In the case of EcoRI, the plasmid can anneal with the presence of foreign DNA via the route of sticky-end ligation, or with "blunt ends" via blunt-end ligation, in the presence of the phage T4 ligase , which forms covalent links between 3-carbon OH and 5-carbon PO4 groups present on blunt ends. Both sticky-end, or overhang ligation and blunt-end ligation can occur between foreign DNA segments, and cleaved ends of the original plasmid depending upon the restriction endonuclease used for cleavage

DNA Microarray Animation

A DNA microarray (also commonly known as gene or genome chip, DNA chip, or gene array) is a collection of microscopic DNA spots, commonly representing single genes, arrayed on a solid surface by covalent attachment to chemically suitable matrices. DNA arrays are different from other types of microarray only in that they either measure DNA or use DNA as part of its detection system. Qualitative or quantitative measurements with DNA microarrays utilize the selective nature of DNA-DNA or DNA-RNA hybridization under high-stringency conditions and fluorophore-based detection. DNA arrays are commonly used for expression profiling, i.e., monitoring expression levels of thousands of genes simultaneously, or for comparative genomic hybridization.

Arrays of DNA can either be spatially arranged, as in the commonly known gene or genome chip, DNA chip, or gene array, or can be specific DNA sequences tagged or labelled such that they can be independently identified in solution. The traditional solid-phase array is a collection of microscopic DNA spots attached to a solid surface, such as glass, plastic or silicon chip. The affixed DNA segments are known as probes (although some sources will use different nomenclature such as reporters), thousands of which can be placed in known locations on a single DNA microarray. Microarray technology evolved from Southern blotting, whereby fragmented DNA is attached to a substrate and then probed with a known gene or fragment.

Applications of these arrays include:

• mRNA or gene expression profiling - Monitoring expression levels for thousands of genes simultaneously is relevant to many areas of biology and medicine, such as studying treatments, disease, and developmental stages. For example, microarrays can be used to identify disease genes by comparing gene expression in diseased and normal cells .
  

• comparative genomic hybridization (Array CGH) - Assessing large genomic rearrangements.
  

• SNP detection arrays - Looking for Single nucleotide polymorphism in the genome of populations.
  

• Chromatin immunoprecipitation (chIP) studies - Determining protein binding site occupancy throughout the genome, employing ChIP-on-chip technology.
  

Fabrication

Microarrays can be fabricated using a variety of technologies, including printing with fine-pointed pins onto glass slides, photolithography using pre-made masks, photolithography using dynamic micromirror devices, ink-jet printing, or electrochemistry on microelectrode arrays.

DNA microarrays can be used to detect RNAs that may or may not be translated into active proteins. Scientists refer to this kind of analysis as "expression analysis" or expression profiling. Since there can be tens of thousands of distinct probes on an array, each microarray experiment can accomplish the equivalent number of genetic tests in parallel. Arrays have therefore dramatically accelerated many types of investigations.

Spotted microarrays
In spotted microarrays (or two-channel or two-colour microarrays), the probes are oligonucleotides, cDNA or small fragments of PCR products that correspond to mRNAs and are spotted onto the microarray surface. This type of array is typically hybridized with cDNA from two samples to be compared (e.g. diseased tissue versus healthy tissue) that are labeled with two different fluorophores (e.g. Rhodamine (Cyanine 5, red) and Fluorescein (Cyanine 3, green)). The two samples are mixed and hybridized to a single microarray that is then scanned in a microarray scanner to visualize fluorescence of the two fluorophores. Relative intensities of each fluorophore are then used to identify up-regulated and down-regulated genes in ratio-based analysis. Absolute levels of gene expression cannot be determined in the two-colour array, but relative differences in expression among different spots (=genes) can be estimated with some oligonucleotide array.


Oligonucleotide microarrays

Two Affymetrix chips

In oligonucleotide microarrays (or single-channel microarrays), the probes are designed to match parts of the sequence of known or predicted mRNAs. T. These microarrays give estimations of the absolute value of gene expression and therefore the comparison of two conditions requires the use of two separate microarrays.

Oligonucleotide Arrays can be either produced by piezoelectric deposition with full length oligonucleotides or in-situ synthesis.

Long Oligonucleotide Arrays are composed of 60-mers, or 50-mers and are produced by ink-jet printing on a silica substrate. Short Oligonucleotide Arrays are composed of 25-mer or 30-mer and are produced by photolithographic synthesis on a silica substrate or piezoelectric deposition on an acrylamide matrix. More recently, Maskless Array Synthesis from NimbleGen Systems has combined flexibility with large numbers of probes. Arrays can contain up to 390,000 spots, from a custom array design. New array formats are being developed to study specific pathways or disease states for a systems biology approach.

Oligonucleotide microarrays often contain control probes designed to hybridize with RNA spike-ins. The degree of hybridization between the spike-ins and the control probes is used to normalize the hybridization measurements for the target probes.


Genotyping microarrays

DNA microarrays can also be used to read the sequence of a genome in particular positions.

SNP microarrays are a particular type of DNA microarrays that are used to identify genetic variation in individuals and across populations. Short oligonucleotide arrays can be used to identify the single nucleotide polymorphisms (SNPs) that are thought to be responsible for genetic variation and the source of susceptibility to genetically caused diseases. Generally termed genotyping applications, DNA microarrays may be used in this fashion for forensic applications, rapidly discovering or measuring genetic predisposition to disease, or identifying DNA-based drug candidates.

These SNP microarrays are also being used to profile somatic mutations in cancer, specifically loss of heterozygosity events and amplifications and deletions of regions of DNA. Amplifications and deletions can also be detected using comparative genomic hybridization, or aCGH, in conjunction with microarrays, but may be limited in detecting novel Copy Number Polymorphisms, or CNPs, by probe coverage.

Resequencing arrays have also been developed to sequence portions of the genome in individuals. These arrays may be used to evaluate germline mutations in individuals, or somatic mutations in cancers.

Genome tiling arrays include overlapping oligonucleotides designed to blanket an entire genomic region of interest. Many companies have successfully designed tiling arrays that cover whole human chromosomes.