Ebola Virus are generally approximately 80 nm in diameter, 970 nm long.
They are cylindrical/tubular, and contain viral envelope, matrix, and nucleocapsid components.
The virus generally appears in a long, filamentous form, but it can also be “U-shaped,” in the shape of a “6” (the “shepherd’s crook” appearance), or even circular.
They have a virally encoded glycoprotein (GP) projecting as 7-10 nm long spikes from its lipid bilayer surface.
Glycoproteins are proteins that contain carbohydrate chains (glycans) covalently attached to their polypeptide side chains, a process known as glycosylation.
The glycoprotein GP is the sole resident of the Ebolavirus surface and is responsible for attaching to and entering new host cells.
The outer viral envelope of the virion is derived by budding from domains of host cell membrane into which the GP spikes have been inserted during their biosynthesis.
This virus belongs to the Filovirus family, and structurally it resembles a length of thread.
Schematic representation of MHC class I MHC class I molecules are one of two primary classes of major histocompatibility complex (MHC) molecules (the other being MHC class II) and are found on nearly every nucleated cell of the body. Their function is to display fragments of proteins from within the cell to T cells; healthy cells will be ignored, while cells containing foreign proteins will be attacked by the immune system. Because MHC class I molecules present peptides derived from cytosolic proteins, the pathway of MHC class I presentation is often called the cytosolic or endogenous pathway.
Contents 1 Function 2 Structure 3 Production 4 Translocation and peptide loading 5 Peptide removal 6 Effect of viruses 7 Genes and isotypes 8 Additional images 9 References 10 External links Function Class I MHC molecules bind peptides generated mainly from degradation of cytosolic proteins by the proteasome. The MHC I:peptide complex is then inserted into the plasma membrane of the cell. The peptide is bound to the extracellular part of the class I MHC molecule. Thus, the function of the class I MHC is to display intracellular proteins to cytotoxic T cells (CTLs). However, class I MHC can also present peptides generated from exogenous proteins, in a process known as cross-presentation.
A normal cell will display peptides from normal cellular protein turnover on its class I MHC, and CTLs will not be activated in response to them due to central and peripheral tolerance mechanisms. When a cell expresses foreign proteins, such as after viral infection, a fraction of the class I MHC will display these peptides on the cell surface. Consequently, CTLs specific for the MHC:peptide complex will recognize and kill the presenting cell.
Alternatively, class I MHC itself can serve as an inhibitory ligand for natural killer cells (NKs). Reduction in the normal levels of surface class I MHC, a mechanism employed by some viruses during immune evasion or in certain tumors, will activate NK cell killing.
Structure MHC class I molecules consist of two polypeptide chains, α and β2-microglobulin (b2m). The two chains are linked noncovalently via interaction of b2m and the α3 domain. Only the α chain is polymorphic and encoded by a HLA gene, while the b2m subunit is not polymorphic and encoded by the Beta-2 microglobulin gene. The α3 domain is plasma membrane-spanning and interacts with the CD8 co-receptor of T-cells. The α1 and α2 domains fold to make up a groove for peptides to bind. MHC class I molecules bind peptides that are 8-10 amino acid in length
Production
Simplified diagram of cytoplasmic protein degradation by the proteasome, transport into endoplasmic reticulum by TAP complex, loading on MHC class I, and transport to the surface for presentation The peptides are generated mainly in the cytosol by the proteasome. The proteasome is a macromolecule that consists of 28 subunits, of which half affect proteolytic activity. The proteasome degrades intracellular proteins into small peptides that are then released into the cytosol. The peptides have to be translocated from the cytosol into the endoplasmic reticulum (ER) to meet the MHC class I molecule, whose peptide-binding site is in the lumen of the ER. They have membrane proximal Ig fold.
Translocation and peptide loading The peptide translocation from the cytosol into the lumen of the ER is accomplished by the transporter associated with antigen processing (TAP). TAP is a member of the ABC transporter family and is a heterodimeric multimembrane-spanning polypeptide consisting of TAP1 and TAP2. The two subunits form a peptide binding site and two ATP binding sites that face the cytosol. TAP binds peptides on the cytoplasmic side and translocates them under ATP consumption into the lumen of the ER. The MHC class I molecule is then, in turn, loaded with peptides in the lumen of the ER.
The peptide-loading process involves several other molecules that form a large multimeric complex consisting of TAP, tapasin, calreticulin, calnexin, and Erp57. Calnexin acts to stabilize the class I MHC α chains prior to β2m binding. Following complete assembly of the MHC molecule, calnexin dissociates. The MHC molecule lacking a bound peptide is inherently unstable and requires the binding of the chaperones calreticulin and Erp57. Additionally, tapasin binds to the MHC molecule and serves to link it to the TAP proteins, thus facilitating enhanced peptide loading and colocalization.
Once the peptide is loaded onto the MHC class I molecule, the complex dissociates and it leaves the ER through the secretory pathway to reach the cell surface. The transport of the MHC class I molecules through the secretory pathway involves several posttranslational modifications of the MHC molecule. Some of the posttranslational modifications occur in the ER and involve change to the N-glycan regions of the protein, followed by extensive changes to the N-glycans in the Golgi apparatus. The N-glycans mature fully before they reach the cell surface.
Peptide removal Peptides that fail to bind MHC class I molecules in the lumen of the endoplasmic reticulum (ER) are removed from the ER via the sec61 channel into the cytosol, where they might undergo further trimming in size, and might be translocated by TAP back into ER for binding to an MHC class I molecule.
For example, an interaction of sec61 with bovine albumin has been observed.
Effect of viruses MHC class I molecules are loaded with peptides generated from the degradation of ubiquitinated cytosolic proteins in proteasomes. As viruses induce cellular expression of viral proteins, some of these products are tagged for degradation, with the resulting peptide fragments entering the endoplasmic reticulum and binding to MHC I molecules. It is in this way, the MHC class I-dependent pathway of antigen presentation, that the virus infected cells signal T-cells that abnormal proteins are being produced as a result of infection.
The fate of the virus-infected cell is almost always induction of apoptosis through cell-mediated immunity, reducing the risk of infecting neighboring cells. As an evolutionary response to this method of immune surveillance, many viruses are able to down-regulate or otherwise prevent the presentation of MHC class I molecules on the cell surface. In contrast to cytotoxic T lymphocytes, Natural killer (NK) cells are normally inactivated upon recognizing MHC I molecules on the surface of cells. Therefore, in the absence of MHC I molecules, NK cells are activated and recognize the cell as aberrant, suggesting they may be infected by viruses attempting to evade immune destruction. Several human cancers also show down-regulation of MHC I, giving transformed cells the same survival advantage of being able to avoid normal immune surveillance designed to destroy any infected or transformed cells.
Electron transport chain COMPLEX 2 Succinate dehydrogenase
Complex II In Complex II (succinate dehydrogenase) additional electrons are delivered into the quinone pool (Q) originating from succinate and transferred (via FAD) to Q. Complex II consists of four protein subunits: SDHA, SDHB, SDHC, and SDHD. Other electron donors (e.g., fatty acids and glycerol 3-phosphate) also direct electrons into Q (via FAD). Complex 2 is a parallel electron transport pathway to complex 1, but unlike complex 1, no protons are transported to the intermembrane space in this pathway. Therefore, the pathway through complex 2 contributes less energy to the overall electron transport chain process.
Electron transport chain COMPLEX 3 cytochrome c reductase
In Complex III (cytochrome bc1 complex; , the Q-cycle contributes to the proton gradient by an asymmetric absorption/release of protons. Two electrons are removed from QH2 at the QO site and sequentially transferred to two molecules of cytochrome c, a water-soluble electron carrier located within the intermembrane space. The two other electrons sequentially pass across the protein to the Qi site where the quinone part of ubiquinone is reduced to quinol. A proton gradient is formed by one quinol (2H+2e-) oxidations at the Qo site to form one quinol (2H+2e-) at the Qi site. (in total six protons are translocated: two protons reduce quinone to quinol and two protons are released from two ubiquinol molecules).
QH2 + 2 cytochrome c (FeIII) + 2 H+in → Q + 2 cytochrome c (FeII) + 4 H+out
When electron transfer is reduced (by a high membrane potential or respiratory inhibitors such as antimycin A), Complex III may leak electrons to molecular oxygen, resulting in superoxide formation.
In secondary active transport, also known as coupled transport or co-transport, energy is used to transport molecules across a membrane; however, in contrast to primary active transport, there is no direct coupling of ATP; instead it relies upon the electrochemical potential difference created by pumping ions in/out of the cell.Permitting one ion or molecule to move down an electrochemical gradient, but possibly against the concentration gradient where it is more concentrated to that where it is less concentrated increases entropy and can serve as a source of energy for metabolism (e.g. in ATP synthase).
In August 1960, in Prague, Robert K. Crane presented for the first time his discovery of the sodium-glucose cotransport as the mechanism for intestinal glucose absorption.Crane's discovery of cotransport was the first ever proposal of flux coupling in biology.
Cotransporters can be classified as symporters and antiporters depending on whether the substances move in the same or opposite directions.
Antiport
Function of Symports and Antiports
In an antiport two species of ion or other solutes are pumped in opposite directions across a membrane. One of these species is allowed to flow from high to low concentration which yields the entropic energy to drive the transport of the other solute from a low concentration region to a high one. An example is the sodium-calcium exchanger or antiporter, which allows three sodium ions into the cell to transport one calcium out.
Many cells also possess a calcium ATPase, which can operate at lower intracellular concentrations of calcium and sets the normal or resting concentration of this important second messenger. But the ATPase exports calcium ions more slowly: only 30 per second versus 2000 per second by the exchanger. The exchanger comes into service when the calcium concentration rises steeply or "spikes" and enables rapid recovery. This shows that a single type of ion can be transported by several enzymes, which need not be active all the time (constitutively), but may exist to meet specific, intermittent needs.
Symport
Symport uses the downhill movement of one solute species from high to low concentration to move another molecule uphill from low concentration to high concentration (against its electrochemical gradient). Both molecules are transported in the same direction.
An example is the glucose symporter SGLT1, which co-transports one glucose (or galactose) molecule into the cell for every two sodium ions it imports into the cell. This symporter is located in the small intestines, trachea, heart, brain, testis, and prostate. It is also located in the S3 segment of the proximal tubule in each nephron in the kidneys.Its mechanism is exploited in glucose rehydration therapy and defects in SGLT1 prevent effective reabsorption of glucose, causing familial renal glucosuria.
Translation is the communication of the meaning of a source-language text by means of an equivalent target-language text.Whereas interpreting undoubtedly antedates writing, translation began only after the appearance of written literature; there exist partial translations of the Sumerian Epic of Gilgamesh (ca. 2000 BCE) into Southwest Asian languages of the second millennium BCE.
Translators always risk inappropriate spill-over of source-language idiom and usage into the target-language translation. On the other hand, spill-overs have imported useful source-language calques and loanwords that have enriched the target languages. Indeed, translators have helped substantially to shape the languages into which they have translated.
Due to the demands of business documentation consequent to the Industrial Revolution that began in the mid-18th century, some translation specialties have become formalized, with dedicated schools and professional associations.
Because of the laboriousness of translation, since the 1940s engineers have sought to automate translation (machine translation) or to mechanically aid the human translator (computer-assisted translation).The rise of the Internet has fostered a world-wide market for translation services and has facilitated language localization.
Translation studies deal with the systematic study of the theory, the description and the application of translation.
Transcription is the first step of gene expression, in which a particular segment of DNA is copied into RNA by the enzyme RNA polymerase. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language. The two can be converted back and forth from DNA to RNA by the action of the correct enzymes. During transcription, a DNA sequence is read by an RNA polymerase, which produces a complementary, antiparallel RNA strand called a primary transcript. Transcription proceeds in the following general steps: One or more sigma factor protein binds to the RNA polymerase holoenzyme, allowing it to bind to promoter DNA. RNA polymerase creates a transcription bubble, which separates the two strands of the DNA helix. This is done by breaking the hydrogen bonds between complementary DNA nucleotides. RNA polymerase adds matching RNA nucleotides to the complementary nucleotides of one DNA strand. RNA sugar-phosphate backbone forms with assistance from RNA polymerase to form an RNA strand. Hydrogen bonds of the untwisted RNA-DNA helix break, freeing the newly synthesized RNA strand. If the cell has a nucleus, the RNA may be further processed. This may include polyadenylation, capping, and splicing. The RNA may remain in the nucleus or exit to the cytoplasm through the nuclear pore complex. The stretch of DNA transcribed into an RNA molecule is called a transcription unit and encodes at least one gene. If the gene transcribed encodes a protein, messenger RNA (mRNA) will be transcribed; the mRNA will in turn serve as a template for the protein's synthesis through translation. Alternatively, the transcribed gene may encode for either non-coding RNA (such as microRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), or other enzymatic RNA molecules called ribozymes. Overall, RNA helps synthesize, regulate, and process proteins; it therefore plays a fundamental role in performing functions within a cell. In virology, the term may also be used when referring to mRNA synthesis from a RNA molecule (i.e., RNA replication). For instance, the genome of an negative-sense single-stranded RNA (ssRNA -) virus may be template a positive-sense single-stranded RNA (ssRNA +). This is since the positive-sense strand contains the information needed to translate the viral proteins for viral replication afterwards. This process is catalysed by a viral RNA replicase.
When a bacterial infection is established in the body, the purpose of the immune system is to control or eradicate it. The initial reaction of the immune system to an infection varies, depending on the site which has been invaded and on the nature of the invader. There can be many "triggers", that can spur the immune system into action. Here are some of the ways in which the immune system can be activated.
If the invasion is in an area of the body that is primarily defended bymacrophages, such as the lungs or intestines, then these macrophages will be the first immune cells on the scene. They begin to digest the invading organism, and by presenting antigens (proteins from the destroyed bacteria), they stimulate other cells of the immune system into action.
Some bacteria, for example Staphylococcus Aureus and Salmonella Typhi, produce chemotaxins when they enter the body, which betray their presence to the immune system, by acting as "breadcrumbs" which reveal the location of the invader. Chemotaxins are chemicals that activatephagocytes, the immune cells whose function it is to consume and destroy the invading bacteria.
Some bacteria first encounter, and are recognised by, the complement system, which in turn produces chemical messengers (cytokines) that warn other cells of the immune system that the body has been invaded.
The invader may be recognised by the acquired immune system, i.e. thelymphocytes. These cells either directly fight the infection themselves, or control other cells to do so.
Immune Cascade
All of the above methods have the effect of inducing phagocytes to migrate to the site of invasion. Once they reach that site, the phagocytes are activated and begin their task of digesting and destroying the invading bacteria. Once activated, they also produce more cytokines, which further activate other cells of the immune system. In a sense, the initial immune reaction leads to a cascade of further immune reactions. Below is a list of the chemical signals that are produced, which cells produce them and the effects that they have on the metabolism.
Cytokines involved in the Inflammatory Response
Cytokine
Producing cell
Action
Interleukin-1
Macrophages
Stimulation of various cells, e.g. T cells, acts to initiate inflammation, induces hypothalamus to increase body temperature
Interleukin-2
T cells
Causes proliferation of activated T and B cells, induces antibody synthesis
Interleukin-3
T cells
Induces growth and differentiation of immune cells in bone marrow
Interleukin-4
T cells
Promotes B cell growth and differentiation
Interleukin-5
T cells
Induces differentiation of B cells, and activates some Microphages
Interleukin-6
T cells, Macrophages
Costimulator of T cells, induces growth in B cells
Interleukin-10
T cells
Activates B cells and inhibits Macrophage function
Interleukin-12
Macrophages
Activates T cells and NK cells
Interleukin-13
T cells
Induces proliferation of B cells and differentiation of T cells
Gamma-Interferon
T cells, NK cells
Activates Macrophages
Tumor Necrosis Factor
Macrophages
Causes activation of some Microphages. Induces inflammation and fever. Induces catabolism of muscle and fat, thus leading to cachexia (bodily wasting)
Transforming Growth Factor
T cells, Macrophages
Inhibits T cell growth and Macrophage activation
Lymphotoxin
T cells
Similar to TNF, activates Microphages
Histamine
Mast cells
Not actually a cytokine, but an important chemical mediator that induces blood vessel dilation and increases cell wall permeability
As you can see from the list above, the cells of the immune system communicate and co-operate in a complex fashion. The full operation of the immune system is far from understood. An important point to note is that invading organisms, if they interfere with any of the chemicals above, or the cells that produce them, can cause a profound change on the bodies immune response to that organism.
Effects of the inflammatory response.
The primary physical effect of the inflammatory response is for blood circulation to increase around the infected area. In particular, the blood vessels around the site of inflammation dilate, permitting increased blood flow to the area. Gaps appear in the cell walls surrounding the infected area, allowing the larger cells of the blood, i.e. the immune cells, to pass. As a result of the increased blood flow, the immune presence is strengthened. All of the different types of cells that constitute the immune system congregate at the site of inflammation, along with a large supply of proteins, which fuel the immune response. There is an increase in body heat, which can itself have an anti-biotic effect, swinging the balance of chemical reactions in favour of the host. The main symptoms of the inflammatory response are as follows.
The tissues in the area are red and warm, as a result of the large amount of blood reaching the site.
The tissues in the area are swollen, again due to the increased amount of blood and proteins that are present.
The area is painful, due the expansion of tissues, causing mechanical pressure on nerve cells, and also due to the presence of pain mediators.
Once the inflammatory process has begun, it continues until the infection that caused it has been eradicated. Phagocytes continue to consume and destroy bacteria, the acquired immune system binds and disposes of harmful toxins. Pus is produced, pus being the debris that is left over from the battle between the invader and the immune system. The colour of the pus depends on the organism causing the infection.
How does the inflammatory response end?
Ideally, the inflammatory response should only last for as long as the infection exists. Once the threat of infection has passed, the area should return to normal existence.
The actual process by which the inflammatory response ends is now only beginning to be understood. The key element is a phenomenon known as "Apoptosis".
When cells of the body die in a normal fashion, e.g. by being irreparably damaged or by being deprived of nutrients, this is known as Necrotic death. Recently, research has shown that cells can also be killed in another way, i.e. by "committing suicide". On receipt of a certain chemical signal, most cells of the body can destroy themselves. This is known as Apoptotic death. There are two main ways in which cells can commit Apoptosis.
By receiving an Apoptosis signal. When an chemical signal is received that indicates that the cell should kill itself, it does so.
By not receiving a "stay-alive" signal. Certain cells, once they reach an activated state, are primed to kill themselves automatically within a certain period of time, i.e. to commit Apoptosis, unless instructed otherwise. However, there may be other cells that supply them with a "stay-alive" signal, which delays the Apoptosis of the cell. It is only when the primed cell stops receiving this "stay-alive" signal that it kills itself.
The immune system employs method two above. The immune cells involved in the inflammatory response, once they become activated, are primed to commitApoptosis. Helper T cells emit a stay-alive signal, and keep emitting that signal for as long as they recognise foreign antigens in the body, prolonging the inflammatory response. It is only when the infection has been eradicated, and there is no more foreign antigen that the helper T cells stop emitting the stay-alive signal, thus allowing the cells involved in the inflammatory response to die off.
If foreign antigen is not eradicated from the body, or the helper T cells do not recognise that fact, or if the immune cells receive the stay-alive signal from another source, then chronic inflammation may develop.
An electron transport chain (ETC) is a series of compounds that transfer electrons from electron donors to electron acceptors via redox reactions, and couples this electron transfer with the transfer of protons (H+ ions) across a membrane. This creates an electrochemical proton gradient that drives ATP synthesis, or the generation of chemical energy in the form of adenosine triphosphate (ATP). Final acceptor of electrons in electron transport chain is molecular oxygen.
Electron transport chains are used for extracting energy via redox reactions from sunlight in photosynthesis or, such as in the case of the oxidation of sugars, cellular respiration. In eukaryotes, an important electron transport chain is found in the inner mitochondrial membrane where it serves as the site of oxidative phosphorylation through the use of ATP synthase. It is also found in the thylakoid membrane of the chloroplast in photosynthetic eukaryotes. In bacteria, the electron transport chain is located in their cell membrane.
In chloroplasts, light drives the conversion of water to oxygen and NADP+ to NADPH with transfer of H+ ions across chloroplast membranes. In mitochondria, it is the conversion of oxygen to water, NADH to NAD+ and succinate to fumarate that are required to generate the proton gradient.
Electron transport chains are major sites of premature electron leakage to oxygen, generating superoxide and potentially resulting in increased oxidative stress.
Mechanism of Transposition-Explainattion/Animation(3D)
Mechanism of transposition in prokaryotes
Several different mechanisms of transposition are employed by prokaryotic transposable elements. And, as we shall see later, eukaryotic elements exhibit still additional mechanisms of transposition.
In E. coli, we can identify replicative and conservative (nonreplicative) modes of transposition. In the replicative pathway, a new copy of the transposable element is generated in the transposition event. The results of the transposition are that one copy appears at the new site and one copy remains at the old site. In the conservative pathway, there is no replication. Instead, the element is excised from the chromosome or plasmid and is integrated into the new site.
When transposition is from one locus to a second locus for certain transposons, a copy of the transposable element is left behind at the first locus. An analysis of transposon mutants revealed an interesting fact about the mechanism of transposition. Using the transposon Tn3 (Figure 20-22), researchers grouped the mutations that prevent transposition into two categories. A trans-recessive class maps in the gene that encodes the transposase enzyme, a catalyst of transposition. A second class of cis-dominant mutations results in the buildup of an intermediate in the transposition process.Figure 20-23 diagrams the transposition pathway in the Tn3 transposition from one plasmid to another. The intermediate is a double plasmid, with both donor and recipient plasmid being fused together. The combined circle resulting from the fusion of two circular elements is termed acointegrate. Apparently, the mutations in this second class delete a region on the transposon at which a recombination event takes place that resolves cointegrates into two smaller circles. This region, called the internal resolution site (IRS), appears in Figure 20-22.
The structure of Tn3. Tn3 contains 4957 base pairs and encodes three polypeptides: the transposase is required for transposition, the repressor is a protein that regulates the transposase gene (see Chapter 11), and β-lactamase confers ampicillin (more...)
Transposition of Tn3 takes place through a cointegrate intermediate. Cointegrates in Tn3 transposition are observed for some internal deletions in the transposon. The correct explanation for this observation is that the cointegrates are intermediates (more...)
The finding of a cointegrate structure as an intermediate in transposition helped establish a replicativemode of transposition for certain elements. In Figure 20-23, note how the transposable element is duplicated in the fusion event and how the recombination event that resolves the cointegrate into two smaller circles leaves one copy of the transposable element in each plasmid.
Some transposons, such as Tn10, excise from the chromosome and integrate into the target DNA. In these cases, DNA replication of the element does not occur, and the element is lost from the site of the original chromosome. Researchers demonstrated this lack of replication by constructing heteroduplexes of λTn10 derivatives containing the lac region of E. coli. The researchers used DNA from Tn10-lacZ+ and Tn10-lacZ− derivatives. The heteroduplexes, therefore, contain one strand with the wild-type lac region and a second strand with the mutated (Z−) lac region. Figure 20-24 diagrams this part of the experiment. The heteroduplex DNA is used to infect cells that have no lac genes, and transpositions of the TetR Tn10 are selected. Different types of colonies arise from the transpositionof a heteroduplex Z−/Z+ carrying transposon (Figure 20-25). If replication takes place (the replicativemode of transposition), all colonies are either completely Lac+ or completely Lac−, because the replication will convert the heteroduplex DNA into two homoduplex daughter molecules. The mechanism by which this conversion takes place will be examined in detail in the next section. However, if the transposition is conservative and does not include replication, each colony arises from a lacZ+/lacZ− heteroduplex. Such colonies are partly Lac+ and partly Lac−. By using media that stain Lac+ and Lac− cells different colors, researchers can observe the Lac+ and Lac− sectors in colonies.
Generation of heteroduplex and homoduplex Tn10 elements. The denaturation and reannealing of a mixture of two parental λ phages carrying Tn10 elements that differ only at three single bases in the transposon yields a mixture of heteroduplex and(more...)
Consequences of conservative and replicative transposition. (a) The heteroduplex or homoduplex nature of DNA (see Figure 20-24) is transposed into a target gene. If the starting DNA is heteroduplex, then the resulting DNA will still be heteroduplex only (more...)
Therefore, the determination of whether Tn10 undergoes replicative or conservative transposition can be made by observing whether differently colored sectors exist within the same colony resulting from the transposition. Sectored colonies are observed in a majority of cases (Figure 20-26). Thus, Tn10—and perhaps other transposable elements in E. coli—transpose by excising themselves from the donor DNA and integrating directly into the recipient DNA.
Colonies resulting from the experiment illustrated in Figures 20-24 and 20-25. A dye is used that stains Z+ cells blue. One-half of this colony is Z+ (dark area), and the other is Z− (white). (Photograph courtesy of N. Kleckner.)
The molecular consequences of transposition reveal an additional piece of evidence concerning the mechanism of transposition: on integration into a new target site, transposable elements generate a repeated sequence of the target DNA in both replicative and conservative transposition. Figure 20-27depicts the integration of IS1 into a gene. In the example shown, the integration event results in the repetition of a 9-bp target sequence. Analysis of many integration events reveals that the repeated sequence does not result from reciprocal site-specific recombination (as is the case in phage λ integration; see page 229); rather, it is generated in the process of integration itself. The number of base pairs is a characteristic of each element. In bacteria, 9-bp and 5-bp repeats are most common.
Duplication of a short sequence of nucleotides in the recipient DNA is associated with the insertion of a transposable element; the two copies bracket the inserted element. Here the duplication that attends the insertion of IS1 is illustrated in a way (more...)
The preceding observations have been incorporated into somewhat complicated models oftransposition. Most models postulate that staggered cleavages are made at the target site and at the ends of the transposable element by a transposase enzyme that is encoded by the element. One end of the transposable element is then attached by a single strand to each protruding end of the staggered cut. Subsequent steps depend on the mode of transposition (replicative or conservative).
Transposable elements generate a high incidence of deletions in their vicinity. These deletions emanate from one end of the element into the surrounding DNA (Figure 20-28). Such events, as well as element-induced inversions, can be viewed as aberrant transposition events. Transposons also give rise to readily detectable deletions in which part of the element is deleted together with varying lengths of the surrounding DNA. This process of imprecise excision is now recognized as deletions or inversions emanating from the internal ends of the IR segments of the transposon. The process ofprecise excision—the loss of the transposable element and the restoration of the gene that was disrupted by the insertion—also occurs, although at very low rates compared with the frequencies of the events just described.
Figure 20-28
Deletion formation mediated by a transposable element. In this example, the transposable element IS1 is shown at a point in theE. coli chromosome near the gal genes. Deletions can be generated from each end of the IS1 element, extending into the neighboring (more...)
MESSAGE
Some DNA sequences in bacteria and phages act as mobile genetic elements. They are capable of joining different pieces of DNA and are thus capable of splicing DNA fragments into or out of the middle of a DNA molecule. Some naturally occurring mobile or transposable elements carry antibiotic-resistance genes.
The basic repeating structural (and functional) unit of chromatin is the nucleosome, which contains nine histone proteins and about 166 base pairs of DNA (Van Holde, 1988; Wolffe, 1999). The observation by electron microscopists that chromatin appeared similar to beads on a string provided an early clue that nucleosomes exist (Olins and Olins, 1974; Woodcock et al., 1976). Another clue came from chemically cross-linking (i.e., joining) histones in chromatin (Thomas & Kornberg, 1975). This experiment demonstrated that H2A, H2B, H3, and H4 form a discrete protein octamer, which is fully consistent with the presence of a repeating histone-containing unit in the chromatin fiber.
Today, researchers know that nucleosomes are structured as follows: Two each of the histones H2A, H2B, H3, and H4 come together to form a histone octamer, which binds and wraps about 1.7 turns of DNA, or about 146 base pairs. The addition of one H1 protein wraps another 20 base pairs, resulting in two full turns around the octamer (Figure 1). Obviously, 166 base pairs are not very much, as each chromosome contains over 100 million base pairs of DNA on average. Therefore, every chromosome contains hundreds of thousands of nucleosomes, and these nucleosomes are joined by the DNA that runs between them (an average of about 20 base pairs). This joining DNA is referred to as linker DNA. Each chromosome is thus a long chain of nucleosomes, which gives the appearance of a string of beads when viewed using an electron microscope (Figure 2; Olins & Olins, 1974, 2003).
A digital model shows the atomic structure of a nucleosome bound to a DNA molecule, from a top-down perspective and in profile against a black background. In the top-down image, at left, a green and brown DNA double-helix forms a closed circle around a complex of blue, green, yellow, and red coiled ribbons. Each ribbon represents a histone protein. In the profile image, at right, the double-helix is wound around the histone proteins in an upside down U-shape.
View Full-Size ImageFigure 3
The amount of DNA per nucleosome was determined by treating chromatin with an enzyme that cuts DNA (such enzymes are called DNases). One such enzyme, micrococcal nuclease (MNase), has the important property of preferentially cutting the linker DNA between nucleosomes well before it cuts the DNA that is wrapped around octamers. By regulating the amount of cutting that occurs after application of MNase, it is possible to stop the reaction before every linker DNA has been cleaved. At this point, the treated chromatin will consist of mononucleosomes, dinucleosomes (connected by linker DNA), trinucleosomes, and so forth (Hewish and Burgoyne, 1973).If DNA from MNase-treated chromatin is then separated on a gel, a number of bands will appear, each having a length that is a multiple of mononucleosomal DNA (Noll, 1974). The simplest explanation for this observation is that chromatin possesses a fundamental repeating structure. When this was considered together with data from electron microscopy and chemical cross-linking of histones, the "subunit theory" of chromatin (Kornberg, 1974; Van Holde et al., 1974) was adopted. The subunits were later named nucleosomes (Oudet et al., 1975) and were eventually crystallized (Luger et al., 1997). The model of the nucleosome that crystallographers constructed from their data is shown in Figure 3. Phosphodiester backbones of the DNA double helix are shown in brown and turquoise, while histone proteins are shown in blue (H3), green (H4), yellow (H2A), and red (H2B). Note that only eukaryotes (i.e., organisms with a nucleus and nuclear envelope) have nucleosomes. Prokaryotes, such as bacteria, do not.
Chromatin Is Coiled into Higher-Order Structures
A black-and-white electron micrograph shows a 30-nanometer chromatin fiber. The fiber appears as a black, vertical, elongated tube against a light grey background. It starts in the upper left, curves toward the right, and then curves back and ends at the center bottom. Other short black tubes appear in the upper right and left middle and bottom.
View Full-Size ImageFigure 4
The packaging of DNA into nucleosomes shortens the fiber length about sevenfold. In other words, a piece of DNA that is 1 meter long will become a "string-of-beads" chromatin fiber just 14 centimeters (about 6 inches) long. Despite this shortening, a half-foot of chromatin is still much too long to fit into the nucleus, which is typically only 10 to 20 microns in diameter. Therefore, chromatin is further coiled into an even shorter, thicker fiber (Figure 4), termed the "30-nanometer fiber," because it is approximately 30 nanometers in diameter. Over the years, there has been a great deal of speculation concerning the manner in which nucleosomes are folded into 30-nanometer fibers (Woodcock, 2005). Part of the problem lies in the fact that electron microscopy is perhaps the best way to visualize packaging, but individual nucleosomes are hard to discern after the fiber has formed. In addition, it also makes a difference whether observations are made using isolated chromatin fibers or chromatin within whole nuclei. Thus, the 30-nanometer fiber may be highly irregular and not quite the uniform structure depicted in textbooks (Figure 1; Bednar et al., 1998). Interestingly, histone H1 is very important in stabilizing chromatin higher-order structures, and 30-nanometer fibers form most readily when H1 is present.
Processes such as transcription and replication require the two strands of DNA to come apart temporarily, thus allowing polymerases access to the DNA template. However, the presence of nucleosomes and the folding of chromatin into 30-nanometer fibers pose barriers to the enzymes that unwind and copy DNA. It is therefore important for cells to have means of opening up chromatin fibers and/or removing histones transiently to permit transcription and replication to proceed. Generally speaking, there are two major mechanisms by which chromatin is made more accessible:
Histones can be enzymatically modified by the addition of acetyl, methyl, or phosphate groups (Fischle et al., 2005).
Histones can be displaced by chromatin remodeling complexes, thereby exposing underlying DNA sequences to polymerases and other enzymes (Smith & Peterson, 2005).
It is important to remember that these processes are reversible, so modified or remodeled chromatin can be returned to its compact state after transcription and/or replication are complete.
Chromosomes Are Most Compacted During Metaphase
When eukaryotic cells divide, genomic DNA must be equally partitioned into both daughter cells. To accomplish this, the DNA becomes highly compacted into the classic metaphase chromosomes that can be seen with a light microscope. Once a cell has divided, its chromosomes uncoil again.
Comparing the length of metaphase chromosomes to that of naked DNA, the packing ratio of DNA in metaphase chromosomes is approximately 10,000:1 (depending on the chromosome). This can be thought of as akin to taking a rope as long as a football field and compacting it down to less than half an inch. This level of compaction is achieved by repeatedly folding chromatin fibers into a hierarchy of multiple loops and coils (Figure 1). Exactly how this is accomplished is unclear, but the phosphorylation of histone H1 may play a role. Indeed, this is just one area of DNA packaging that researchers will continue to explore in the years to come.
Methyl directed mismatch repair HD Animation-Explainattion/Animation(3D)
DNA mismatch repair is a system for recognizing and repairing erroneous insertion, deletion, and mis-incorporation of bases that can arise during DNA replication and recombination, as well as repairing some forms of DNA damage.
Mismatch repair is strand-specific. During DNA synthesis the newly synthesised (daughter) strand will commonly include errors. In order to begin repair, the mismatch repair machinery distinguishes the newly synthesised strand from the template (parental). In gram-negative bacteria, transient hemimethylation distinguishes the strands (the parental is methylated and daughter is not). However, in other prokaryotes and eukaryotes, the exact mechanism is not clear. It is suspected that, in eukaryotes, newly synthesized lagging-strand DNA transiently contains nicks (before being sealed by DNA ligase) and provides a signal that directs mismatch proofreading systems to the appropriate strand. This implies that these nicks must be present in the leading strand, and evidence for this has recently been found.[3] Recent work [4] has shown that nicks are sites for RFC-dependent loading of the replication sliding clamp PCNA, in an orientation-specific manner, such that one face of the donut-shape protein is juxtaposed toward the 3'-OH end at the nick. Oriented PCNA then directs the action of the MutLalpha endonuclease to one strand in the presence of a mismatch and MutSalpha or MutSbeta.
Any mutational event that disrupts the superhelical structure of DNA carries with it the potential to compromise the genetic stability of a cell. The fact that the damage detection and repair systems are as complex as the replication machinery itself highlights the importance evolution has attached to DNA fidelity.
Examples of mismatched bases include a G/T or A/C pairing (see DNA repair). Mismatches are commonly due to tautomerization of bases during G2. The damage is repaired by recognition of the deformity caused by the mismatch, determining the template and non-template strand, and excising the wrongly incorporated base and replacing it with the correct nucleotide. The removal process involves more than just the mismatched nucleotide itself. A few or up to thousands of base pairs of the newly synthesized DNA strand can be removed.
DNA mismatch repair (MMR) is an evolutionarily conserved process that corrects mismatches generated during DNA replication and escape proofreading. MMR proteins also participate in many other DNA transactions, such that inactivation of MMR can have wide-ranging biological consequences, which can be either beneficial or detrimental. We begin this review by briefly considering the multiple functions of MMR proteins and the consequences of impaired function. We then focus on the biochemical mechanism of MMR replication errors. Emphasis is on structure-function studies of MMR proteins, on how mismatches are recognized, on the process by which the newly replicated strand is identified, and on excision of the replication error.
