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.

COMPARATIVE GENOMICS LECTURE

Comparative genomics is the study of relationships between the genomes of different species or strains. Comparative genomics is an attempt to take advantage of the information provided by the signatures of selection to understand the function and evolutionary processes that act on genomes. While it is still a young field, it holds great promise to yield insights into many aspects of the evolution of modern species. The sheer amount of information contained in modern genomes (several gigabytes in the case of humans) necessitates that the methods of comparative genomics are mostly computational in nature. Gene finding is an important application of comparative genomics, as is discovery of new, non-coding functional elements of the genome.

Comparative genomics exploits both similarities and differences in the proteins, RNA, and regulatory regions of different organisms to infer how selection has acted upon these elements. Those elements that are responsible for similarities between different species should be conserved through time (stabilizing selection), while those elements responsible for differences among species should be divergent (positive selection). Finally, those elements that are unimportant to the evolutionary success of the organism will be unconserved (selection is neutral).


Identifying the mechanisms of eukaryotic genome evolution by comparative genomics is one of the important goals of the field. It is however often complicated by the multiplicity of events that have taken place throughout the history of individual lineages, leaving only distorted and superimposed traces in the genome of each living organism. For this reason comparative genomics studies of small model organisms (for example yeast) are of great importance to advance our understanding of general mechanisms of evolution.

Having come a long way from its initial use of finding functional proteins, comparative genomics is now concentrating on finding regulatory regions and siRNA molecules. Recently, it has been discovered that distantly related species often share long conserved stretches of DNA that do not appear to code for any protein. It is unknown at this time what function such ultra-conserved regions serve.

Immune system Animation

An immune system is a collection of mechanisms within an organism that protects against infection by identifying and killing pathogens. It detects pathogens ranging from viruses to parasitic worms and distinguishes them from the organism's normal cells and tissues. Detection is complicated as pathogens adapt and evolve new ways to successfully infect the host organism.

To survive this challenge, several mechanisms have evolved that recognize and neutralize pathogens. Even simple unicellular organisms such as bacteria possess enzyme systems that protect against viral infections. Other basic immune mechanisms evolved in ancient eukaryotes and remain in their modern descendants, such as plants, fish, reptiles, and insects. These mechanisms include antimicrobial peptides called defensins, pattern recognition receptors, and the complement system. More sophisticated mechanisms, however, developed relatively recently, with the evolution of vertebrates.The immune systems of vertebrates such as humans consist of many types of proteins, cells, organs, and tissues, which interact in an elaborate and dynamic network. As part of this more complex immune response, the vertebrate system adapts over time to recognize particular pathogens more efficiently. The adaptation process creates immunological memories and allows even more effective protection during future encounters with these pathogens. This process of acquired immunity is the basis of vaccination.

Disorders in the immune system can cause disease. Immunodeficiency diseases occur when the immune system is less active than normal, resulting in recurring and life-threatening infections. Immunodeficiency can either be the result of a genetic disease, such as severe combined immunodeficiency, or be produced by pharmaceuticals or an infection, such as the acquired immune deficiency syndrome (AIDS) that is caused by the retrovirus HIV. In contrast, autoimmune diseases result from a hyperactive immune system attacking normal tissues as if they were foreign organisms. Common autoimmune diseases include rheumatoid arthritis, diabetes mellitus type 1 and lupus erythematosus. These critical roles of immunology in health and disease are areas of intense scientific study.



Layered defense in immunity

The immune system protects organisms from infection with layered defenses of increasing specificity. Most simply, physical barriers prevent pathogens such as bacteria and viruses from entering the body. If a pathogen breaches these barriers, the innate immune system provides an immediate, but non-specific response. Innate immune systems are found in all plants and animals. However, if pathogens successfully evade the innate response, vertebrates possess a third layer of protection, the adaptive immune system. Here, the immune system adapts its response during an infection to improve its recognition of the pathogen. This improved response is then retained after the pathogen has been eliminated, in the form of an immunological memory, and allows the adaptive immune system to mount faster and stronger attacks each time this pathogen is encountered.

Surface barriers

Several barriers protect organisms from infection, including mechanical, chemical and biological barriers. The waxy cuticle of many leaves, the exoskeleton of insects, the shells and membranes of externally deposited eggs, and skin are examples of the mechanical barriers that are the first line of defense against infection.
However, as organisms cannot be completely sealed against their environments, other systems act to protect body openings such as the lungs, intestines, and the genitourinary tract. In the lungs, coughing and sneezing mechanically eject pathogens and other irritants from the respiratory tract. The flushing action of tears and urine also mechanically expels pathogens, while mucus secreted by the respiratory and gastrointestinal tract serves to trap and entangle microorganisms.

Chemical barriers also protect against infection. The skin and respiratory tract secrete antimicrobial peptides such as the β-defensins. Enzymes such as lysozyme and phospholipase A in saliva, tears, and breast milk are also antibacterials. Vaginal secretions serve as a chemical barrier following menarche, when they become slightly acidic, while semen contains defensins and zinc to kill pathogens. In the stomach, gastric acid and proteases serve as powerful chemical defenses against ingested pathogens.

Within the genitourinary and gastrointestinal tracts, commensal flora serve as biological barriers by competing with pathogenic bacteria for food and space and, in some cases, by changing the conditions in their environment, such as pH or available iron.This reduces the probability that pathogens will be able to reach sufficient numbers to cause illness. However, since most antibiotics non-specifically target bacteria and do not affect fungi, oral antibiotics can lead to an “overgrowth” of fungi and cause conditions such as a vaginal candidiasis (yeast infection). There is good evidence that re-introduction of probiotic flora, such as pure cultures of the lactobacilli normally found in yoghurt, helps restore a healthy balance of microbial populations in intestinal infections in children and encouraging preliminary data in studies on bacterial gastroenteritis, inflammatory bowel diseases, urinary tract infection and post-surgical infections.

Innate immunity

Microorganisms that successfully enter an organism will encounter the cells and mechanisms of the innate immune system. Innate immune defenses are non-specific, meaning these systems recognize and respond to pathogens in a generic way. This system does not confer long-lasting immunity against a pathogen. The innate immune system is the dominant system of host defense in most organisms

Humoral and chemical barriers

Inflammation

Inflammation is one of the first responses of the immune system to infection. The symptoms of inflammation are redness and swelling, which are caused by increased blood flow into a tissue. Inflammation is produced by eicosanoids and cytokines, which are released by injured or infected cells. Eicosanoids include prostaglandins that produce fever and the dilation of blood vessels associated with inflammation, and leukotrienes that attract certain white blood cells (leukocytes). Common cytokines include interleukins that are responsible for communication between white blood cells; chemokines that promote chemotaxis; and interferons that have anti-viral effects, such as shutting down protein synthesis in the host cell. Growth factors and cytotoxic factors may also be released. These cytokines and other chemicals recruit immune cells to the site of infection and promote healing of any damaged tissue following the removal of pathogens.

Complement system

he complement system is a biochemical cascade that attacks the surfaces of foreign cells. It contains over 20 different proteins and is named for its ability to “complement” the killing of pathogens by antibodies. Complement is the major humoral component of the innate immune response. Many species have complement systems, including non-mammals like plants, fish, and some invertebrates.

In humans, this response is activated by the binding of complement proteins to carbohydrates on the surfaces of microbes or by complement binding to antibodies that have attached to these microbes. This recognition signal triggers a rapid killing response. The speed of the response is a result of signal amplification that occurs following sequential proteolytic activation of complement molecules, which are also proteases. After complement proteins initially bind to the microbe, they activate their protease activity, which in turn activates other complement proteases, and so on. This produces a catalytic cascade that amplifies the initial signal by controlled positive feedback. The cascade results in the production of peptides that attract immune cells, increase vascular permeability, and opsonize (coat) the surface of a pathogen, marking it for destruction. This deposition of complement can also kill cells directly by disrupting their plasma membrane.

Adaptive immunity
The adaptive immune system evolved in early vertebrates and allows for a stronger immune response as well as immunological memory, where each pathogen is "remembered" by a signature antigen.The adaptive immune response is antigen-specific and requires the recognition of specific “non-self” antigens during a process called antigen presentation. Antigen specificity allows for the generation of responses that are tailored to specific pathogens or pathogen-infected cells. The ability to mount these tailored responses is maintained in the body by "memory cells". Should a pathogen infect the body more than once, these specific memory cells are used to quickly eliminate it.

Lymphocytes

The cells of the adaptive immune system are special types of leukocytes, called lymphocytes. B cells and T cells are the major types of lymphocytes and are derived from hematopoietic stem cells in the bone marrow.B cells are involved in the humoral immune response, whereas T cells are involved in cell-mediated immune response.

Association of a T cell with MHC class I or MHC class II, and antigen (in red)

Both B cells and T cells carry receptor molecules that recognize specific targets. T cells recognize a “non-self” target, such as a pathogen, only after antigens (small fragments of the pathogen) have been processed and presented in combination with a “self” receptor called a major histocompatibility complex (MHC) molecule. There are two major subtypes of T cells: the killer T cell and the helper T cell. Killer T cells only recognize antigens coupled to Class I MHC molecules, while helper T cells only recognize antigens coupled to Class II MHC molecules. These two mechanisms of antigen presentation reflect the different roles of the two types of T cell. A third, minor subtype are the γδ T cells that recognize intact antigens that are not bound to MHC receptors.

In contrast, the B cell antigen-specific receptor is an antibody molecule on the B cell surface, and recognizes whole pathogens without any need for antigen processing. Each lineage of B cell expresses a different antibody, so the complete set of B cell antigen receptors represent all the antibodies that the body can manufacture.


Killer T cells

Killer T cell are a sub-group of T cells that kill cells infected with viruses (and other pathogens), or are otherwise damaged or dysfunctional. As with B cells, each type of T cell recognises a different antigen. Killer T cells are activated when their T cell receptor (TCR) binds to this specific antigen in a complex with the MHC Class I receptor of another cell. Recognition of this MHC:antigen complex is aided by a co-receptor on the T cell, called CD8. The T cell then travels throughout the body in search of cells where the MHC I receptors bear this antigen. When an activated T cell contacts such cells, it releases cytotoxins that form pores in the target cell's plasma membrane, allowing ions, water and toxins to enter. This causes the target cell to undergo apoptosis. T cell killing of host cells is particularly important in preventing the replication of viruses. T cell activation is tightly controlled and generally requires a very strong MHC/antigen activation signal, or additional activation signals provided by "helper" T cells.

Helper T cells

Helper T cells regulate both the innate and adaptive immune responses and help determine which types of immune responses the body will make to a particular pathogen. These cells have no cytotoxic activity and do not kill infected cells or clear pathogens directly. They instead control the immune response by directing other cells to perform these tasks.

Helper T cells express T cell receptors (TCR) that recognize antigen bound to Class II MHC molecules. The MHC:antigen complex is also recognized by the helper cell's CD4 co-receptor, which recruits molecules inside the T cell (e.g. Lck) that are responsible for T cell's activation. Helper T cells have a weaker association with the MHC:antigen complex than observed for killer T cells, meaning many receptors (around 200–300) on the helper T cell must be bound by an MHC:antigen in order to activate the helper cell, while killer T cells can be activated by engagement of a single MHC:antigen molecule. Helper T cell activation also requires longer duration of engagement with an antigen-presenting cell. The activation of a resting helper T cell causes it to release cytokines that influence the activity of many cell types. Cytokine signals produced by helper T cells enhance the microbicidal function of macrophages and the activity of killer T cells. In addition, helper T cell activation causes an upregulation of molecules expressed on the T cell's surface, such as CD40 ligand (also called CD154), which provide extra stimulatory signals typically required to activate antibody-producing B cells.

Meiosis Animation

In biology, meiosis (IPA: /maɪˈəʊsɪs/) is the process by which one diploid eukaryotic cell divides to generate four haploid cells often called gametes. The word "meiosis" comes from the Greek meioun, meaning "to make smaller," since it results in a reduction in chromosome number in the gamete cell. Among fungi, spores in which the haploid nuclei are at first disseminated are called meiospores, or more specifically, ascospores in asci (Ascomycota) and basidospores on basidia (Basidiomycota).

Meiosis is essential for sexual reproduction and therefore occurs in all eukaryotes, including single-celled organisms that reproduce sexually. A few eukaryotes, notably the Bdelloid rotifers, have lost the ability to carry out meiosis and have acquired the ability to reproduce by parthenogenesis. Meiosis does not occur in archaea or prokaryotes, which reproduce by asexual mitotic cell division.

Meiosis uses many of the same biochemical mechanisms employed during mitosis to accomplish the redistribution of chromosomes. There are several features unique to meiosis, most importantly the pairing and genetic recombination between homologous chromosomes

During meiosis, the genome of a diploid germ cell, which is composed of long segments of DNA packaged into chromosomes, undergoes DNA replication followed by two rounds of division, resulting in haploid cells called gametes. Each gamete contains one complete set of chromosomes, or half of the genetic content of the original cell. These resultant haploid cells can fuse with other haploid cells of the opposite sex or mating type during fertilization to create a new diploid cell, or zygote. Thus, the division mechanism of meiosis is a reciprocal process to the joining of two genomes that occurs at fertilization. Because the chromosomes of each parent undergo genetic recombination during meiosis, each gamete, and thus each zygote, will have a unique genetic blueprint encoded in its DNA. In other words, meiosis and sexual reproduction produce genetic variation.

Occurrence of meiosis in eukaryotic life cycles

Meiosis occurs in all eukaryotic life cycles involving sexual reproduction, comprising of the constant cyclical process of meiosis and fertilization. This takes place alongside normal mitotic cell division. In multicellular organisms, there is an intermediary step between the diploid and haploid transition where the organism grows. The organism will then produce the germ cells that continue in the life cycle. The rest of the cells, called somatic cells, function within the organism and will die with it.

The organism phase of the life cycle can occur between the haploid to diploid transition or the diploid to haploid transition. Some species are diploid, grown from a diploid cell called the zygote. Others are haploid instead, spawned by the proliferation and differentiation of a single haploid cell called the gamete. Humans, for example, are diploid creatures. Human stem cells undergo meiosis to create haploid gametes, which are spermatozoa for males or ova for females. These gametes then fertilize in the Fallopian tubes of the female, producing a diploid zygote. The zygote undergoes progressive stages of mitosis and differentiation, turns into a blastocyst and then gets implanted in the uterus endometrium to create an embryo.

There are three types of life cycles that utilize sexual reproduction, differentiated by the location of the organisms stage.

In the gametic life cycle, of which humans are a part, the living organism is diploid in nature. Here, we will generalize the example of human reproduction stated previously. The organism's diploid germ-line stem cells undergo meiosis to create haploid gametes, which fertilize to form the zygote. The diploid zygote undergoes repeated cellular division by mitosis to grow into the organism. Mitosis is a related process to meiosis that creates two cells that are genetically identical to the parent cell. The general principle is that mitosis creates somatic cells and meiosis creates germ cells.

In the zygotic life cycle, the living organism is haploid. Two organisms of opposing gender contribute their haploid germ cells to form a diploid zygote. The zygote undergoes meiosis immediately, creating four haploid cells. These cells undergo mitosis to create the organism. Many fungi and many protozoa are members of the zygotic life cycle.

Finally, in the sporic life cycle, the living organism alternates between haploid and diploid states. Consequently, this cycle is also known as the alternation of generations. The diploid organism's germ-line cells undergo meiosis to produce gametes. The gametes proliferate by mitosis, growing into a haploid organism. The haploid organism's germ cells then combine with another haploid organism's cells, creating the zygote. The zygote undergoes repeated mitosis and differentiation to become the diploid organism again. The sporic life cycle can be considered a fusion of the gametic and zygotic life cycles, and indeed its diagram supports this conclusion.

Process

Because meiosis is a "one-way" process, it cannot be said to engage in a cell cycle as mitosis does. However, the preparatory steps that lead up to meiosis are identical in pattern and name to the interphase of the mitotic cell cycle.

Interphase is divided into three phases:
Growth 1 (G1) phase: Characterized by increase in cell size due to accelerated manufacture of organelles, proteins, and other cellular matter.
Synthesis (S) phase: The genetic material is replicated: each of its chromosomes duplicates. The cell is still diploid, however, because it still contains the same number of centromeres.
Growth 2 (G2) phase: The cell continues to grow.

Interphase is immediately followed by meiosis I and meiosis II. Meiosis I consists of segregating the homologous chromosomes from each other, then dividing the tetraploid cell into two diploid cells each containing one of the segregates. Meiosis II consists of decoupling each chromosome's sister strands (chromatids), segregating the DNA into two sets of strands (each set containing one of each homolog), and dividing both diploid cells to produce four haploid cells. Meiosis I and II are both divided into prophase, metaphase, anaphase, and telophase subphases, similar in purpose to their analogous subphases in the mitotic cell cycle. Therefore, meiosis encompasses the interphase (G1, S, G2), meiosis I (prophase I, metaphase I, anaphase I, telophase I), and meiosis II (prophase II, metaphase II, anaphase II, telophase II).

Meiosis I
Prophase I

The first stage of prophase I is the leptotene stage, also known as leptonema, from Greek words meaning "thin threads." During this stage, individual chromosomes begin to condense into long strands within the nucleus. However the two sister chromatids are still so tightly bound that they are indistinguishable from one another.

The zygotene stage, also known as zygonema, from Greek words meaning "paired threads,
occurs as the chromosomes approximately line up with each other into homologous chromosomes. The combined homologous chromosomes are said to be bivalent. They may also be referred to as a tetrad, a reference to the four sister chromatids. The two chromatids become "zipped" together, forming the synaptonemal complex, in a process known as synapsis.

The pachytene stage, also known as pachynema, from Greek words meaning "thick threads," heralds crossing over. Nonsister chromatids of homologous chromosomes randomly exchange segments of genetic information over regions of homology. (Sex chromosomes, however, are not identical, and only exchange information over a small region of homology.) Exchange takes place at sites where recombination nodules have formed. The exchange of information between the non-sister chromatids results in a recombination of information; each chromosome has the complete set of information it had before, and there are no gaps formed as a result of the process. Because the chromosomes cannot be distinguished in the synaptonemal complex, the actual act of crossing over is not perceivable through the microscope.

During the diplotene stage, also known as diplonema, from Greek words meaning "two threads," the synaptonemal complex degrades and homologous chromosomes separate from one another a little. The chromosomes themselves uncoil a bit, allowing some transcription of DNA. However, the homologous chromosomes of each bivalent remain tightly bound at chiasmata, the regions where crossing over occurred.

Chromosomes condense further during the diakinesis stage, from Greek words meaning "moving through." This is the first point in meiosis where the four parts of the tetrads are actually visible. Sites of crossing over entangle together, effectively overlapping, making chiasmata clearly visible. Other than this observation, the rest of the stage closely resembles prometaphase of mitosis; the nucleoli disappears, the nuclear membrane disintegrates into vesicles, and the mitotic spindle begins to form.

During these stages, centrioles are migrating to the two poles of the cell. These centrioles, which were duplicated during interphase, function as microtubule coordinating centers. Centrioles sprout microtubules, essentially cellular ropes and poles, during crossing over. They invade the nuclear membrane after it disintegrates, attaching to the chromosomes at the kinetochore. The kinetochore functions as a motor, pulling the chromosome along the attached microtubule toward the originating centriole, like a train on a track. There are two kinetochores on each tetrad, one for each centrosome. Prophase I is the longest phase in meiosis.

Microtubules that attach to the kinetochores are known as kinetochore microtubules. Other microtubules will interact with microtubules from the opposite centriole. These are called nonkinetochore microtubules.

Metaphase I

Homologous pairs move together along the phase plate: as kinetochore microtubules from both centrioles attach to their respective kinetochores, the homologous chromosomes align along an equatorial plane that bisects the spindle, due to continuous counterbalancing forces exerted on the bivalents by the microtubules emanating from the two kinetochores. The physical basis of the independent assortment of chromosomes is the random orientation of each bivalent along the metaphase plate.

Anaphase I

Kinetochore microtubules shorten, severing the recombination nodules and pulling homologous chromosomes apart. Since each chromosome only has one kinetochore, whole chromosomes are pulled toward opposing poles, forming two diploid sets. Each chromosome still contains a pair of sister chromatids. Nonkinetochore microtubules lengthen, pushing the centrioles further apart. The cell elongates in preparation for division down the middle. In prophase 1 the DNA coils tightly and individual chromosomes become visible under the light microscope. Homologous chromosomes closely associated in synapsis and they exchange segments by crossing over.

Telophase I

The first meiotic division effectively ends when the centromeres arrive at the poles. Each daughter cell now has half the number of chromosomes but each chromosome consists of a pair of chromatids. This effect produces a variety of responses from the neuro-synrchromatic enzyme, also known as NSE. The microtubules that make up the spindle network disappear, and a new nuclear membrane surrounds each haploid set. The chromosomes uncoil back into chromatin. Cytokinesis, the pinching of the cell membrane in animal cells or the formation of the cell wall in plant cells, occurs, completing the creation of two daughter cells.

Cells enter a period of rest known as interkinesis or interphase II. No DNA replication occurs during this stage. Note that many plants skip telophase I and interphase II, going immediately into prophase II.

Meiosis II

Prophase II takes an inversely proportional time compared to telophase I. In this prophase we see the disappearance of the nucleoli and the nuclear envelope again as well as the shortening and thickening of the chromatids. Centrioles move to the polar regions and are arranged by spindle fibres. The new equatorial plane is rotated by 90 degrees when compared to meiosis I, perpendicular to the previous plane.

In metaphase II, the centromeres contain three kinetochores, organizing fibers from the centrosomes on each side.

This is followed by anaphase II, where the centromeres are cleaved, allowing the kinetochores to pull the sister chromatids apart. The sister chromatids by convention are now called sister chromosomes, and they are pulled toward opposing poles.

The process ends with telophase II, which is similar to telophase I, marked by uncoiling, lengthening, and disappearance of the chromosomes occur as the disappearance of the microtubules. Nuclear envelopes reform; cleavage or cell wall formation eventually produces a total of four daughter cells, each with a haploid set of chromosomes. Meiosis is now complete.

Significance of meiosis
Meiosis facilitates stable sexual reproduction. Without the halving of ploidy, or chromosome count, fertilization would result in zygotes that have twice the number of chromosomes than the zygotes from the previous generation. Successive generations would have an exponential increase in chromosome count, resulting in an unwieldy genome that would cripple the reproductive fitness of the species. Polyploidy, the state of having three or more sets of chromosomes.

Most importantly, however, meiosis produces genetic variety in gametes that propagate to offspring. Recombination and independent assortment allow for a greater diversity of genotypes in the population. As a system of creating diversity, meiosis allows a species to maintain stability under environmental changes.

Nondisjunction
The normal separation of chromosomes in Meiosis I or sister chromatids in meiosis II is termed disjunction. When the separation is not normal, it is called nondisjunction. This results in the production of gametes which have either more or less of the usual amount of genetic material, and is a common mechanism for trisomy or monosomy. Nondisjunction can occur in the meiosis I or meiosis II, phases of cellular reproduction, or during mitosis.

This is a cause of several medical conditions in humans, including:
Down's Syndrome - trisomy of chromosome 21
Patau Syndrome - trisomy of chromosome 13
Edward Syndrome - trisomy of chromosome 18
Klinefelter Syndrome - extra X chromosomes in males - ie XXY, XXXY, XXXXY
Turner Syndrome - atypical X chromosome dosage in in females - ie XO, XXX, XXXX
XYY Syndrome - an extra Y chromosome in males

Meiosis in humans
In females, meiosis occurs in precursor cells known as oogonia that divide twice into oocytes. These stem cells stop at the diplotene stage of meiosis I and lay dormant within a protective shell of somatic cells called the follicle. Follicles begin growth at a steady pace in a process known as folliculogenesis, and a small number enter the menstrual cycle. Menstruated oocytes continue meiosis I and arrest at meiosis II until fertilization. The process of meiosis in females is called oogenesis, and differs from the typical meiosis in that it features a long period of meiotic arrest known as the Dictyate stage and lacks the assistance of centrosomes.

In males, meiosis occurs in precursor cells known as spermatogonia that divide twice to become sperm. These cells continuously divide without arrest in the seminiferous tubules of the testicles. Sperm is produced at a steady pace. The process of meiosis in males is called spermatogenesis.

DNA Replication Animation

DNA replication is the process of copying a double-stranded deoxyribonucleic acid (DNA) molecule, a process essential in all known life forms. The general mechanisms of DNA replication are different in prokaryotic and eukaryotic organisms.

As each DNA strand holds the same genetic information, both strands can serve as templates for the reproduction of the opposite strand. The template strand is preserved in its entirety and the new strand is assembled from nucleotides. This process is called semiconservative replication. The resulting double-stranded DNA molecules are identical; proofreading and error-checking mechanisms exist to ensure extremely high fidelity.

In a cell, DNA replication must happen before cell division. Prokaryotes replicate their DNA throughout the interval between cell divisions. In eukaryotes, timings are highly regulated and this occurs during the S phase of the cell cycle, preceding mitosis or meiosis I.

DNA structure

A DNA strand is a long polymer built from nucleotides; two complementary DNA strands form a double helix, each strand possessing a 5' phosphate end and a 3' hydroxyl end. The numbers followed by the prime indicate the position on the deoxyribose sugar backbone to which the phosphate or hydroxyl group is attached (numbers without primes are reserved for the bases). The two strands in the DNA backbone run anti-parallel to each other: One of the DNA strands is built in the 5' → 3' direction while the other runs in an anti parallel direction, although its information is stored in the 3' → 5' direction. Each nucleotide consists of a phosphate, a simple sugar or a deoxyribose sugar - forming the backbone of the DNA double helix - plus a base. The bonding angles of the backbone ensures that DNA will tend to twist as the length of the molecule progresses, giving rise a double helix shape instead of a straight ladder. Base pairs form the steps of the helix ladder while the sugars and phosphate molecules forms the handrail. Each of the four bases has a partner to which it makes the strongest hydrogen bonds. When a nucleotide base forms hydrogen bonds with its complementary base on the other strand, they form a base pair: Adenine pairs with thymine and cytosine pairs with guanine. These pairings can be expressed as C•G and A•T, or C≡G and A=T where the number of lines indicate the number of hydrogen bonds between each base pair. For example, a 10-base pair strand running in the 5' → 3' direction that has adenine as the 3rd base will pair with the base thymine as the 7th base on the complementary 10-base pair strand running in the opposite direction.

The replication fork

The replication fork is a structure which forms when DNA is being replicated. It is created through the action of helicase, which breaks the hydrogen bonds holding the two DNA strands together. The resulting structure has two branching "prongs", each one made up of a single strand of DNA.

Lagging strand synthesis

In DNA replication, the lagging strand is the DNA strand at the replication fork opposite to the leading strand. It is also oriented in the opposite direction when compared to the leading strand, with the 5' near the replication fork instead of the 3' end as is the case with the leading strand. When the enzyme helicase unwinds DNA, two single stranded regions of DNA (the "replication fork") form. DNA polymerase cannot build a strand in the 3' → 5' direction. This poses no problems for the leading strand, which can continuously synthesize DNA in a processive manner, but creates a problem for the lagging strand, which cannot be synthesized in the 3' → 5' direction. Thus, the lagging strand is synthesized in short segments known as Okazaki fragments. On the lagging strand, primase builds an RNA primer in short bursts. DNA polymerase is then able to use the free 3' hydroxyl group on the RNA primer to synthesize DNA in the 5' → 3' direction. The RNA fragments are then removed (different mechanisms are used in eukaryotes and prokaryotes) and new deoxyribonucleotides are added to fill the gaps where the RNA was present. DNA ligase is then able to join the deoxyribonucleotides together, completing the synthesis of the lagging strand.

Leading strand synthesis

The leading strand is defined as the DNA strand that is read in the 3' → 5' direction but synthesized in the 5'→ 3' direction, in a continuous manner. On this strand, DNA polymerase III is able to synthesize DNA using the free 3'-OH group donated by a single RNA primer (multiple RNA primers are not used) and continuous synthesis occurs in the direction in which the replication fork is moving.

Dynamics at the replication fork

The sliding clamp in all domains of life share a similar structure, and are able to interact with the various processive and non-processive DNA polymerases found in cells. In addition, the sliding clamp serves as a processivity factor. The C-terminal end of the clamps forms loops which are able to interact with other proteins involved in DNA replication (such as DNA polymerase and the clamp loader). The inner face of the clamp allows DNA to be threaded through it. The sliding clamp forms no specific interactions with DNA. There is a large 35A hole in the middle of the clamp. This allows DNA to fit through it, and water to take up the rest of the space allowing the clamp to slide along the DNA. Once the polymerase reaches the end of the template or detects double stranded DNA (see below), the sliding clamp undergoes a conformational change which releases the DNA polymerase.

The clamp loader, a multisubunit protein, is able to bind to the sliding clamp and DNA polymerase. When ATP is hydrolyzed, it loses affinity for the sliding clamp allowing DNA polymerase to bind to it. Furthermore, the sliding clamp can only be bound to a polymerase as long as single stranded DNA is being synthesized. Once the single stranded DNA runs out, the polymerase is able to bind to the a subunit on the clamp loader and move to a new position on the lagging strand. On the leading strand, DNA polymerase III associates with the clamp loader and is bound to the sliding clamp.

Recent evidence suggests that the enzymes and proteins involved in DNA replication remain stationary at the replication forks while DNA is looped out to maintain bidirectionality observed in replication. This is a result of an interaction between DNA polymerase, the sliding clamp, and the clamp loader.

DNA replication

DNA replication differs somewhat between eukaryotic and prokaryotic cells. Much of our knowledge of prokaryotic DNA replication was derived from the study of E. coli, while yeast has been used as a model organism for understanding eukaryotic DNA replication.

Mechanism of replication

Once priming of DNA is complete, DNA polymerase is loaded into the DNA and replication begins. The catalytic mechanism of DNA polymerase involves the use of two metal ions in the active site and a region in the active site that can discriminate between deoxyribonucleotides and ribonucleotides. The metal ions are general divalent cations that help the 3'-OH initiate a nucleophilic attack on the alpha-phosphate of the deoxyribonucleotide and orient and stabilize the negatively charged triphosphate on the deoxyribonucleotide. Nucleophilic attack by the 3'-OH on the alpha phosphate releases pyrophosphate, which is then subsequently hydrolyzed by inorganic pyrophosphatase into two phosphates. This hydrolysis drives DNA synthesis to completion.

Furthermore, DNA polymerase must be able to distinguish between correctly paired bases and incorrectly paired bases. This is accomplished by distinguishing Watson-Crick base pairs through the use of an active site pocket that is complementary in shape to the structure of correctly paired nucleotides. This pocket has a tyrosine residue that is able to form van der Waals interactions with the correctly paired nucleotide. In addition, double stranded DNA in the active site has a wider and shallower minor groove that permits the formation of hydrogen bonds with the third nitrogen of purine bases and the second oxygen of pyrimidine bases. Finally, the active site makes extensive hydrogen bonds with the DNA backbone. These interactions result in the DNA polymerase III closing around a correctly paired base. If a base is inserted and incorrectly paired, these interactions could not occur due to disruptions in hydrogen bonding and van der Waals interactions. The mechanism of replication is similar in eukaryotes and prokaryotes.

DNA is read in the 3' → 5' direction, relative to the parent strand, therefore, nucleotides are synthesized (or attached to the template strand) in the 5' → 3' direction, relative to the daughter strand. However, one of the parent strands of DNA is 3' → 5' and the other is 5' → 3'. To solve this, replication must proceed in opposite directions. The leading strand runs towards the replication fork and is thus synthesized in a continuous fashion, only requiring one primer. On the other hand, the lagging strand runs in the opposite direction, heading away from the replication fork, and is synthesized in a series of short fragments known as Okazaki fragments, consequently requiring many primers. The RNA primers of Okazaki fragments are subsequently degraded by RNase H and DNA Polymerase I (exonuclease), and the gaps (or nicks) are filled with deoxyribonucleotides and sealed by the enzyme ligase.

Diabetes Animation

Diabetes mellitus (IPA pronunciation: [ˌdaɪəˈbitəs]). is a metabolic disorder characterized by hyperglycemia (high blood sugar) and other signs, as distinct from a single disease or condition. The World Health Organization recognizes three main forms of diabetes: type 1, type 2, and gestational diabetes (occurring during pregnancy), which have similar signs, symptoms, and consequences, but different causes and population distributions. Type 1 is usually due to autoimmune destruction of the pancreatic beta cells which produce insulin. Type 2 is characterized by tissue-wide insulin resistance and varies widely; it sometimes progresses to loss of beta cell function. Gestational diabetes is similar to type 2 diabetes, in that it involves insulin resistance; the hormones of pregnancy cause insulin resistance in those women genetically predisposed to developing this condition.

Type 1 diabetes mellitus—formerly known as insulin-dependent diabetes (IDDM), childhood diabetes or also known as juvenile diabetes, is characterized by loss of the insulin-producing beta cells of the islets of Langerhans of the pancreas leading to a deficiency of insulin. It should be noted that there is no known preventative measure that can be taken against type 1 diabetes. Most people affected by type 1 diabetes are otherwise healthy and of a healthy weight when onset occurs. Diet and exercise cannot reverse or prevent type 1 diabetes. Sensitivity and responsiveness to insulin are usually normal, especially in the early stages. This type comprises up to 10% of total cases in North America and Europe, though this varies by geographical location. This type of diabetes can affect children or adults but was traditionally termed "juvenile diabetes" because it represents a majority of cases of diabetes affecting children.

The most common cause of beta cell loss leading to type 1 diabetes is autoimmune destruction, accompanied by antibodies directed against insulin and islet cell proteins. The principal treatment of type 1 diabetes, even from the earliest stages, is replacement of insulin. Without insulin, ketosis and diabetic ketoacidosis can develop and coma or death will result.

Currently, type 1 diabetes can be treated only with insulin, with careful monitoring of blood glucose levels using blood testing monitors. Emphasis is also placed on lifestyle adjustments (diet and exercise). Apart from the common subcutaneous injections, it is also possible to deliver insulin by a pump, which allows continuous infusion of insulin 24 hours a day at preset levels and the ability to program doses (a bolus) of insulin as needed at meal times. It is also possible to deliver insulin with an inhaled powder.

Type 1 treatment must be continued indefinitely. Treatment does not impair normal activities, if sufficient awareness, appropriate care, and discipline in testing and medication is taken. The average glucose level for the type 1 patient should be as close to normal (80–120 mg/dl, 4–6 mmol/l) as possible. Some physicians suggest up to 140–150 mg/dl (7-7.5 mmol/l) for those having trouble with lower values, such as frequent hypoglycemic events. Values above 200 mg/dl (10 mmol/l) are often accompanied by discomfort and frequent urination leading to dehydration. Values above 300 mg/dl (15 mmol/l) usually require immediate treatment and may lead to ketoacidosis. Low levels of blood glucose, called hypoglycemia, may lead to seizures or episodes of unconsciousness.

Type 2 diabetes mellitus—previously known as adult-onset diabetes, maturity-onset diabetes, or non-insulin-dependent diabetes mellitus (NIDDM)—is due to a combination of defective insulin secretion and insulin resistance or reduced insulin sensitivity (defective responsiveness of tissues to insulin), which almost certainly involves the insulin receptor in cell membranes. In the early stage the predominant abnormality is reduced insulin sensitivity, characterized by elevated levels of insulin in the blood. At this stage hyperglycemia can be reversed by a variety of measures and medications that improve insulin sensitivity or reduce glucose production by the liver, but as the disease progresses the impairment of insulin secretion worsens, and therapeutic replacement of insulin often becomes necessary. There are numerous theories as to the exact cause and mechanism for this resistance, but central obesity (fat concentrated around the waist in relation to abdominal organs, and not subcutaneous fat, it seems) is known to predispose individuals for insulin resistance, possibly due to its secretion of adipokines (a group of hormones) that impair glucose tolerance. Abdominal fat is especially active hormonally. Obesity is found in approximately 55% of patients diagnosed with type 2 diabetes. Other factors include aging (about 20% of elderly patients are diabetic in North America) and family history (Type 2 is much more common in those with close relatives who have had it), although in the last decade it has increasingly begun to affect children and adolescents, likely in connection with the greatly increased childhood obesity seen in recent decades in some places.

Type 2 diabetes may go unnoticed for years in a patient before diagnosis, as visible symptoms are typically mild or non-existent, without ketoacidotic episodes, and can be sporadic as well. However, severe long-term complications can result from unnoticed type 2 diabetes, including renal failure, vascular disease (including coronary artery disease), vision damage, etc.

Type 2 diabetes is usually first treated by attempts to change physical activity (generally an increase is desired), the diet (generally to decrease carbohydrate intake), and weight loss. These can restore insulin sensitivity, even when the weight loss is modest, for example, around 5 kg (10 to 15 lb), most especially when it is in abdominal fat deposits. Some Type 2 diabetics can achieve satisfactory glucose control, sometimes for years, as a result. However, the underlying tendency to insulin resistance is not lost, and so attention to diet, exercise, and weight loss must continue. The usual next step, if necessary, is treatment with oral antidiabetic drugs. As insulin production is initially unimpaired in Type 2s, oral medication (often used in various combinations) can still be used to improve insulin production (e.g., sulfonylureas), to regulate inappropriate release of glucose by the liver (and attenuate insulin resistance to some extent (e.g., metformin), and to substantially attenuate insulin resistance (e.g., thiazolidinediones). According to one study, overweight patients treated with metformin compared with diet alone, had relative risk reductions of 32% for any diabetes endpoint, 42% for diabetes related death and 36% for all cause mortality and stroke. When oral medications fail (cessation of beta cell insulin secretion is not uncommon amongst Type 2s), insulin therapy will be necessary to maintain normal or near normal glucose levels. A disciplined regimen of blood glucose checks is recommended in most cases, most particularly and necessarily when taking medications.