Genetic recombination is a process by which a molecule of nucleic acid (usually DNA, but can also be RNA) is broken and

then joined to a different one. Recombination can occur between similar molecules of DNA, as in homologous recombination, or dissimilar molecules, as in nonhomologous end joining. Recombination is a common method of DNA repair in both bacteria and eukaryotes. In eukaryotes, recombination also occurs in meiosis, where it facilitates chromosomal crossover. The crossover process leads to offspring's having different combinations of genes from those of their parents, and can occasionally produce new chimericalleles. In organisms with an adaptive immune system, a type of genetic recombination called V(D)J recombination helps immune cells rapidly diversify to recognize and adapt to new pathogens. The shuffling of genes brought about by genetic recombination is thought to have many advantages, as it is a major engine of genetic variation and also allows sexually reproducing organisms to avoid Muller's ratchet, in which the genomes of an asexualpopulation accumulate deleterious mutations in an irreversible manner. In genetic engineering, recombination can also refer to artificial and deliberate recombination of disparate pieces of DNA, often from different organisms, creating what is called recombinant DNA. A prime example of such a use of genetic recombination is gene targeting, which can be used to add, delete or otherwise change an organism's genes. This technique is important to biomedical researchers as it allows them to study the effects of specific genes. Techniques based on genetic recombination are also applied in protein engineering to develop new proteins of biological interest. Genetic recombination is catalyzed by many different enzymes, called recombinases. RecA, the chief recombinase found in Escherichia coli, is responsible for the repair of DNA double strand breaks (DSBs). In yeast and other eukaryotic organisms there are two recombinases required for repairing DSBs. The RAD51 protein is required for mitotic and meiotic recombination, whereas the DMC1 protein is specific to meiotic recombination.

Chromosomal crossover

Thomas Hunt Morgan's illustration of crossing over (1916) Chromosomal crossover refers to recombination between the paired chromosomes inherited from each of one's parents, generally occurring during meiosis. During prophase I the four available chromatids are in tight formation with one another. While in this formation, homologous sites on two chromatids can mesh with one another, and may exchange genetic information.[1]

Because recombination can occur with small probability at any location along chromosome, the frequency of recombination between two locations depends on their distance. Therefore, for genes sufficiently distant on the same chromosome the amount of crossover is high enough to destroy the correlation between alleles. Tracking the movement of genes during crossovers has proven quite useful to geneticists. Because two genes that are close together are less likely to become separated than genes that are farther apart, geneticists can deduce roughly how far apart two genes are on a chromosome if they know the frequency of the crossovers. Geneticists can also use this method to infer the presence of certain genes. Genes that typically stay together during recombination are said to be

linked. One gene in a linked pair can sometimes be used as a marker to deduce the presence of another gene. This is typically used in order to detect the presence of a disease-causing gene.[2]

Gene conversion

The difference between gene conversion and chromosomal crossover. Blue are the two chromatids of one chromosome and red are the two chromatids of another one. In gene conversion, a section of genetic material is copied from one chromosome to another, without the donating chromosome being changed. Gene conversion occurs at high frequency during meiotic division and at low frequency in somatic cells. It is a process by which a DNA sequence is copied from one DNA helix (which remains unchanged) to another DNA helix, whose sequence is altered. It is one of the ways a gene may be mutated. Gene conversion may lead to non-Mendelian inheritance and has often been recorded in fungal crosses.[3]

Nonhomologous recombination
Recombination can occur between DNA sequences that contain no sequence homology. This is referred to as nonhomologous recombination or nonhomologous end joining.[1]
In B cells

Holliday junction

Holliday Junction

Molecular structure of a Holliday junction.From PDB3CRX. A Holliday junction is a mobile junction between four strands of DNA. The structure is named after Robin Holliday, who proposed it in 1964[1][2][3] to account for a particular type of exchange of genetic information he observed in yeast known as homologous recombination. Holliday junctions are highly conserved structures, from prokaryotes to mammals.[4] Because these junctions are between homologous sequences, they can slide up and down the DNA. In bacteria, this sliding (or branch migration) is facilitated by the RuvABC complex or RecG protein, molecular motors that use the energy of ATP hydrolysis to push the junction around. The junction must then be resolved, split up, to restore 2 linear duplexes. This can be done to either restore the parental configuration or to establish a crossed over configuration. Resolution can occur in either a horizontal or vertical fashion during homologous recombination, giving patch products (if in same orientation during double strand break repair) or splice products (if in different orientations during double strand break repair). Holliday junctions are an intermediate in genetic recombination which are also of importance in maintaining genomic integrity.[1][5]

Details
In prophase of meiosis I, duplicated homologous chromosomes pair and align end-to-end. Crossover can occur between aligned chromatids, leading to exchange of homologous segments by homologous recombination. Chromosome segregation through meiotic divisions leads to novel genotypes, first in gametes, then in offspring. In the original Holliday model for homologous recombination, single-strand breaks occur at the same point on one strand of each parental DNA. Free ends of each broken strand then migrate across to the other DNA helix, where the invading strands are joined to the free ends they encounter. The resulting crossover junction is called a Holliday junction. As each crossover strand reanneals to its original partner strand it displaces the original complementary strand ahead of it, causing the Holliday junction to migrate. This creates heteroduplex DNA segments. Cleavage and rejoining to re-establish two separate DNAs occurs in two ways. This is most easily visualized by first rearranging the Holliday structure. DNA molecules with this apparent structure have been observed. This structure can be resolved by cleavage in the horizontal plane, leading to two molecules that do not show crossover of markers in genes A and B. If, instead, the same structure is cleaved in the vertical plane, both of the resulting recombinant molecules show crossover of markers in genes A and B. All products, regardless of cleavage plane, are heteroduplexes in the region of Holliday junction migration.

RecA

Crystal structure of a RecA-DNA complex (PDB ID: 3cmt). A double-strand break and two adjacent 3' overhangs are visible between the two RecA chains.

RecA is a 38 kilodaltonEscherichia coliprotein essential for the repair and maintenance of DNA. RecA has a structural and functional homolog in every species in which it has been seriously sought and serves as an archetype for this class of homologous DNA repair proteins. The homologous protein in Homo sapiens is called RAD51. RecA has multiple activities, all related to DNA repair. In the bacterialSOS response, it has a coprotease function in the autocatalytic cleavage of the LexArepressor and the repressor.[1] RecA's association with DNA major is based on its central role in homologous recombination. The RecA protein binds strongly and in long clusters to ssDNA to form a nucleoprotein filament. The protein has more than one DNA binding site, and thus can hold a single strand and double strand together. This feature makes it possible to catalyze a DNA synapsis reaction between a DNA double helix and a homologous region of single stranded DNA. The reaction initiates the exchange of strands between two recombining DNA double helices. After the synapsis event, in the heteroduplex region a process called branch migration begins. In branch migration an unpaired region of one of the single strands displaces a paired region of the other single strand, moving the branch point without changing the total number of base pairs. Spontaneous branch migration can occur, however as it generally proceeds equally in both directions it is unlikely to complete recombination efficiently. The RecA protein catalyzes unidirectional branch migration and by doing so makes it possible to complete recombination, producing a region of heteroduplex DNA that is thousands of base pairs long.

Since it is a DNA-dependent ATPase, RecA contains an additional site for binding and hydrolyzing ATP. RecAassociates more tightly with DNA when it has ATP bound than when it has ADP bound. E. coli strains deficient in RecA are useful for cloning procedures in molecular biology laboratories. E. coli strains are often genetically modified to contain a mutant recAallele and thereby ensure the stability of extrachromosomal segments of DNA, known as plasmids. In a process called transformation, plasmid DNA is taken up by the bacteria under a variety of conditions. Bacteria containing exogenous plasmids are called "transformants". Transformants retain the plasmid throughout cell divisions such that it can be recovered and used in other applications. Without functional RecA protein, the exogenous plasmid DNA is left unaltered by the bacteria. Purification of this plasmid from bacterial cultures can then allow high-fidelity PCR amplification of the original plasmid sequence.

Potential as a drug target

Wigle and Singleton at the University of North Carolina have shown that small molecules interfering with RecA function in the cell may be useful in the creation of new antibiotic drugs.[2] Since many antibiotics lead to DNA damage, and all bacteria rely on RecA to fix this damage, inhibitors of RecA could be used to enhance the toxicity of antibiotics. Additionally the activities of RecA are synonymous with antibiotic resistance development, and inhibitors of RecA may also serve to delay or prevent the appearance of bacterial drug resistance.

SOS response

The SOS response has been proposed as a model for bacterial evolution of certain types of antibiotic resistance.[1] The SOS response is a global response to DNA damage in which the cell cycle is arrested and DNA repair and mutagenesis are induced. The SOS uses the RecA protein (Rad51 in eukaryotes). The RecA protein, stimulated by single-stranded DNA, is involved in the

inactivation of the LexA repressor thereby inducing the response. It is an error-prone repair system. During normal growth, the SOS genes are negatively regulated by LexArepressor protein dimers. Under normal conditions, LexA binds to a 20-bp consensus sequence (the SOS box) in the operator region for those genes. Some of these SOS genes are expressed at certain levels even in the repressed state, according to the affinity of LexA for their SOS box. Activation of the SOS genes occurs after DNA damage by the accumulation of single stranded (ssDNA) regions generated at replication forks, where DNA polymerase is blocked. RecA forms a filament around these ssDNA regions in an ATP-dependent fashion, and becomes activated. The activated form of RecA interacts with the LexA repressor to facilitate the LexA repressor's self-cleavage from the operator.[2] Once the pool of LexA decreases, repression of the SOS genes goes down according to the level of LexA affinity for the SOS boxes. Operators that bind LexA weakly are the first to be fully expressed. In this way LexA can sequentially activate different mechanisms of repair. Genes having a weak SOS box (such as lexA, recA, uvrA, uvrB, and uvrD) are fully induced in response to even weak SOS-inducing treatments. Thus the first SOS repair mechanism to be induced is nucleotide excision repair (NER), whose aim is to fix DNA damage without commitment to a full-fledged SOS response. If, however, NER does not suffice to fix the damage, the LexA concentration is further reduced, so the expression of genes with stronger LexA boxes (such as sulA, umuD, umuC - these are expressed late) is induced. SulA stops cell division by binding to FtsZ, the initiating protein in this process. This causes filamentation, and the induction of UmuDC-dependent mutagenic repair. As a result of these properties, some genes may be partially induced in response to even endogenous levels of DNA damage, while other genes appear to be induced only when high or persistent DNA damage is present in the cell.

Discovery
The SOS response was discovered and named by MiroslavRadman in 1975.[3]

Antibiotic resistance
Recent research has shown that the SOS pathway may be essential in the acquisition of bacterial mutations which lead to resistance to some antibiotic drugs.[4] The increased rate of mutation during the SOS response is caused by three low-fidelity DNA polymerases: Pol II, Pol IV and Pol V.[4] Researchers are now targeting these proteins with the aim of creating drugs that prevent SOS repair. By doing so, the time needed for pathogenic bacteria to evolve antibiotic resistance could be extended, and thus improve the long term viability of some antibiotic drugs.[5]

Mobile genetic elements

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Mobile genetic elements in the Cell (left) and in what ways they can be acquired (right). Mobile genetic elements (MGE) are a type of DNA that can move around within the genome. They include:
y

y y y

Transposons (also called transposable elements) o Retrotransposons o DNA transposons o Insertion sequences Plasmids Bacteriophage elements, like Mu which integrates randomly into the genome Group II introns

The total of all mobile genetic elements in a genome may be referred to as the mobilome.

Barbara McClintock was awarded the 1983Nobel Prize in Physiology or Medicine "for her discovery of mobile genetic elements".[1]

Transposon
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A DNA composite transposon Transposons are sequences of DNA that can move or transpose themselves to new positions within the genome of a single cell. The mechanism of transposition can be either "copy and paste" or "cut and paste". Transposition can create phenotypically significant mutations and alter the cell's genome size. Barbara McClintock's discovery of these jumping genes early in her career earned her a Nobel prize in 1983.[1] Transposons make up a large fraction of the C-value of eukaryotic cells. Transposons are often considered "junk DNA". In Oxytricha, which has a unique genetic system, they play a critical role in its development.[2] Transposons are very useful to researchers as a means to alter DNA inside a living organism.

Types
Transposons are only one of several types of mobile genetic elements. Transposons themselves are assigned to one of two classes according to their mechanism of transposition, which can be described as either "copy and paste" (class I) or "cut and paste" (class II).[3] Class I (Retrotransposons): They copy themselves in two stages, first from DNA to RNA by transcription, then from RNA back to DNA by reverse transcription. The DNA copy is then inserted into the genome in a new position. Reverse transcription is catalyzed by a reverse transcriptase, which is often coded by the transposon itself. Retrotransposons behave very similarly to retroviruses, such as HIV. There are three main subclasses of retrotransposons:
y y y

Viral: encode reverse transcriptase (to reverse transcribe RNA into DNA), have long terminal repeats (LTRs), similar to retroviruses LINEs: encode reverse transcriptase, lack LTRs, transcribed by RNA polymerase II Nonviral superfamily: do not code for reverse transcriptase, transcribed by RNA polymerase III

Class II (DNA transposons): By contrast, the cut-and-paste transposition mechanisms of class II transposons do not involve an RNA intermediate. These transpositions are catalyzed by various types of transposase enzymes. Some transposases can bind non-specifically to any target site, while others bind to specific sequence targets. The transposase makes a staggered cut at the target site producing sticky ends, cuts out the transposon and ligates it into the target site. A DNA polymerase fills in the resulting gaps from the sticky ends and DNA ligase closes the sugar-phosphate backbone. This results in target site duplication and the insertion sites of DNA transposons may be identified by short direct repeats (a staggered cut in the target DNA filled by DNA polymerase) followed by inverted repeats (which are important for the transposon excision by transposase). The duplications at the target site can result in gene duplication, which plays an important role in evolution[4]:284. Not all DNA transposons transpose through a cut-and-paste mechanism. In some cases a replicative transposition is observed in which transposon replicates itself to a new target site. Cut-and-paste transposons may be duplicated if transposition takes place during S phase of the cell cycle when the "donor" site has already been replicated, but the "target" site has not. Both classes of transposons may lose their ability to synthesise reverse transcriptase or transposase through mutation, yet continue to jump through the genome because other transposons are still producing the necessary enzymes. Hence, DNA transposons can be classified as either "autonomous" or "non-autonomous". Autonomous transposons have an intact gene that encodes an active transposase enzyme; the transposon does not need another source of transposase for its transposition. In contrast, non-autonomous transposons encode defective polypeptides and accordingly require transposase from another source. When a transposon is

used as a genetic tool, the transposase is supplied by the investigator, often from an expression cassette within a plasmid.[5] Retroviruses can be considered as transposable elements: After entering a host cell and converting their RNA into DNA, retroviruses integrate this DNA into the DNA of the host cell. The integrated DNA form (provirus) of the retrovirus is viewed as a particularly specialized form of eukaryoticretrotransposon, which is able to encode RNA intermediates that usually can leave the host cells and infect other cells. The transposition cycle of retroviruses also has similarities to that of prokaryotic transposons. The similarities suggest a distant familial relationship between these two transposon types.

volution
The evolution of transposons and their effect on genome evolution is currently a dynamic field of study. Transposons are found in many major branches of life. They may or may not have originated in the last universal common ancestor, or arisen independently multiple times, or perhaps arisen once and then spread to other kingdoms by horizontal gene transfer.[19] While some transposons may confer benefits on their hosts, most are regarded as selfish DNAparasites. In this way, they are similar to viruses. Various viruses and transposons also share features in their genome structures and biochemical abilities, leading to speculation that they share a common ancestor. Since excessive transposon activity can destroy a genome, many organisms have developed mechanisms to inhibit this activity. Bacteria may undergo high rates of gene deletion as part of a mechanism to remove transposons and viruses from their genomes while eukaryoticorganisms use RNA interference (RNAi) to inhibit transposon activity. Evolution has been particularly harsh on DNA transposons. In vertebrate animal cells nearly all >100,000 DNA transposons per genome have genes that encode inactive transposase polypeptides.[20] In humans, all of the Tc1-like transposons are inactive. As a result the first DNA transposon used as a tool for genetic purposes, the Sleeping Beauty transposon system, was a Tc1/mariner-like transposon that was resurrected from an long evolutionary sleep.[21] Interspersed Repeats within genomes are created by transposition events accumulating over evolutionary time. Because interspersed repeats block gene conversion, they protect novel gene sequences from being overwritten by similar gene sequences and thereby facilitate the development of new genes.

Transposons may have been co-opted by the vertebrate immune system as a means of producing antibody diversity: The V(D)J recombination system operates by a mechanism similar to that of transposons.

[edit] Applications
Main article: Transposons as a genetic tool The first transposon was discovered in the plant maize (Zea mays, corn species), and is named dissociator (Ds). Likewise, the first transposon to be molecularly isolated was from a plant (Snapdragon). Appropriately, transposons have been an especially useful tool in plant molecular biology. Researchers use transposons as a means of mutagenesis. In this context, a transposon jumps into a gene and produces a mutation. The presence of the transposon provides a straightforward means of identifying the mutant allele, relative to chemical mutagenesis methods. Sometimes the insertion of a transposon into a gene can disrupt that gene's function in a reversible manner, in a process called insertional mutagenesis; transposase-mediated excision of the transposon restores gene function. This produces plants in which neighboring cells have different genotypes. This feature allows researchers to distinguish between genes that must be present inside of a cell in order to function (cell-autonomous) and genes that produce observable effects in cells other than those where the gene is expressed. Transposons are also a widely used tool for mutagenesis of most experimentally tractable organisms. The Sleeping Beauty transposon system has been used extensively as an insertional tag for identifying cancer genes [22] The Tc1/mariner-class transposon Sleeping Beauty transposon system, awarded as the Molecule of the Year 2009[23] is active in mammalian cells and are being investigated for use in human gene therapy.[24

Plasmid

Figure 1: Illustration of a bacterium with plasmid enclosed showing chromosomal DNA and plasmids.

In microbiology and genetics, a plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA.[1] They are double-stranded and, in many cases, circular. Plasmids usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms (e.g., the 2-micrometre-ring in Saccharomyces cerevisiae). Plasmid sizes vary from 1 to over 1,000 kilobase pairs (kbp).[2][3][4] The number of identical plasmids in a single cell can range anywhere from one to even thousands under some circumstances. Plasmids can be considered part of the mobilome because they are often associated with conjugation, a mechanism of horizontal gene transfer. The term plasmid was first introduced by the Americanmolecular biologistJoshua Lederberg in 1952.[5] Plasmids are considered "replicons", capable of autonomous replication within a suitable host. Plasmids can be found in all three major domains: Archea, Bacteria and Eukarya.[1] Similar to viruses, plasmids are not considered by some to be a form of "life".[6] Unlike viruses, plasmids are "naked" DNA and do not encode genes necessary to encase the genetic material for transfer to a new host, though some classes of plasmids encode the sex pilus necessary for their own transfer. Plasmid host-to-host transfer requires direct, mechanical transfer by conjugation or changes in host gene expression allowing the intentional uptake of the genetic element by transformation.[1] Microbial transformation with plasmid DNA is neither parasitic nor symbiotic in nature, because each implies the presence of an independent species living in a commensal or detrimental state with the host organism. Rather, plasmids provide a mechanism for horizontal gene transfer within a population of microbes and typically provide a selective advantage under a given environmental state. Plasmids may carry genes that provide resistance to naturally occurring antibiotics in a competitive environmental niche, or alternatively the proteins produced may act as toxins under similar circumstances. Plasmids can also provide bacteria with the ability to fix elemental nitrogen or to degrade recalcitrant organic compounds which provide an advantage when nutrients are scarce.

Vectors

There are two types of plasmid integration into a host bacteria: Non-integrating plasmids replicate as with the top instance; whereas episomes, the lower example, integrate into the host chromosome. Plasmids used in genetic engineering are called vectors. Plasmids serve as important tools in genetics and biotechnology labs, where they are commonly used to multiply (make many copies of) or express particular genes.[2] Many plasmids are commercially available for such uses. The gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics and a multiple cloning site (MCS, or polylinker), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this location. Next, the plasmids are inserted into bacteria by a process called transformation. Then, the bacteria are exposed to the particular antibiotics. Only bacteria which take up copies of the plasmid survive, since the plasmid makes them resistant. In particular, the protecting genes are expressed (used to make a protein) and the expressed protein breaks down the antibiotics. In this way the antibiotics act as a filter to select only the modified bacteria. Now these bacteria can be grown in large amounts, harvested and lysed (often using the alkaline lysis method) to isolate the plasmid of interest. Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. Just as the bacteria produces proteins to confer its antibiotic resistance, it can also be induced to produce large amounts of proteins from the inserted gene. This is a cheap and easy way of mass-producing a gene or the protein it then codes for, for example, insulin or even antibiotics.

However, a plasmid can only contain inserts of about 110 kbp. To clone longer lengths of DNA, lambda phage with lysogeny genes deleted, cosmids, bacterial artificial chromosomes or yeast artificial chromosomes are used.

Applications
Disease models
Plasmids were historically used to genetically engineer the embryonic stem cells of rats in order to create rat genetic disease models. The limited efficiency of plasmid based techniques precluded their use in the creation of more accurate human cell models. Fortunately, developments in Adeno-associated virus recombination techniques, and Zinc finger nucleases, have enabled the creation of a new generation of isogenic human disease models.

Gene therapy
The success of some strategies of gene therapy depends on the efficient insertion of therapeutic genes at the appropriate chromosomal target sites within the human genome, without causing cell injury, oncogenic mutations (cancer) or an immune response. Plasmid vectors are one of many approaches that could be used for this purpose. Zinc finger nucleases (ZFNs) offer a way to cause a site-specific double strand break to the DNA genome and cause homologous recombination. This makes targeted gene correction a possibility in human cells. Plasmids encoding ZFN could be used to deliver a therapeutic gene to a pre-selected chromosomal site with a frequency higher than that of random integration. Although the practicality of this approach to gene therapy has yet to be proven, some aspects of it could be less problematic than the alternative viral-based delivery of therapeutic genes.[7]

Episomes
An episome is a portion of genetic material that can sometimes exist in the cytoplasm, independent of the chromosome, while at other times is integrated into the chromosome.[8] Examples include insertion sequences and transposons. Another example of an episome is called the F factor. The F factor determines whether genetic material in the chromosome of one organism is transferred into another organism. The F factor can exist in three states that are designated as F+, Hfr, and F prime. F+ refers to the F factor that exists independently of the chromosome. Hfr stands for high frequency of recombination, and refers to a factor that has integrated into the host chromosome. The F prime factor exists outside the chromosome, but has a portion of chromosomal DNA attached to it.[citation needed]

Types

Overview of bacterial conjugation

Electron micrograph of a DNA fiber bundle, presumably of a single bacterial chromosome loop.

Electron micrograph of a bacterial DNA plasmid (chromosome fragment). One way of grouping plasmids is by their ability to transfer to other bacteria. Conjugative plasmids contain tra genes, which perform the complex process of conjugation, the transfer of plasmids to another bacterium (Fig. 4). Non-conjugative plasmids are incapable of initiating conjugation, hence they can only be transferred with the assistance of conjugative plasmids. An intermediate class of plasmids aremobilizable, and carry only a subset of the genes required for transfer. They can parasitize a conjugative plasmid, transferring at high frequency only in its presence. Plasmids are now being used to manipulate DNA and may possibly be a tool for curing many diseases. It is possible for plasmids of different types to coexist in a single cell. Several different plasmids have been found in E. coli. However, related plasmids are often incompatible, in the sense that only one of them survives in the cell line, due to the regulation of vital plasmid functions. Therefore, plasmids can be assigned into compatibility groups. Another way to classify plasmids is by function. There are five main classes:
y y

Fertility F-plasmids, which contain tra genes. They are capable of conjugation. Resistance (R)plasmids, which contain genes that can build a resistance against antibiotics or poisons and help bacteria produce pili. Historically known as R-factors, before the nature of plasmids was understood. Col plasmids, which contain genes that code for bacteriocins, proteins that can kill other bacteria.

y y

Degradative plasmids, which enable the digestion of unusual substances, e.g.toluene or salicylic acid. Virulence plasmids, which turn the bacterium into a pathogen.

Plasmids can belong to more than one of these functional groups. Plasmids that exist only as one or a few copies in each bacterium are, upon cell division, in danger of being lost in one of the segregating bacteria. Such single-copy plasmids have systems which attempt to actively distribute a copy to both daughter cells. These systems are often referred to as the partition system or partition function of a plasmid. Some plasmids or microbial hosts include an addiction system or postsegregational killing system (PSK), such as the hok/sok (host killing/suppressor of killing) system of plasmid R1 in Escherichia coli.[9] This variant produces both a long-lived poison and a short-lived antidote. Several types of plasmid addiction systems (toxin/ antitoxin, metabolism-based, ORT systems) were described in the literature[10] and used in biotechnical (fermentation) or biomedical (vaccine therapy) applications. Daughter cells that retain a copy of the plasmid survive, while a daughter cell that fails to inherit the plasmid dies or suffers a reduced growth-rate because of the lingering poison from the parent cell. Finally, the overall productivity could be enhanced.

Yeast plasmids
Other types of plasmids are often related to yeast cloning vectors that include:
y

Yeast integrative plasmid (YIp), yeast vectors that rely on integration into the host chromosome for survival and replication, and are usually used when studying the functionality of a solo gene or when the gene is toxic. Also connected with the gene URA3, that codes an enzyme related to the biosynthesis of pyrimidine nucleotides (T, C); Yeast Replicative Plasmid (YRp), which transport a sequence of chromosomal DNA that includes an origin of replication. These plasmids are less stable, as they can "get lost" during the budding.

Plasmid DNA extraction

As alluded to above, plasmids are often used to purify a specific sequence, since they can easily be purified away from the rest of the genome. For their use as vectors, and for molecular cloning, plasmids often need to be isolated. There are several methods to isolate plasmid DNA from bacteria, the archetypes of which are the miniprep and the maxiprep/bulkprep.[2] The former can be used to quickly find out whether the plasmid is correct in any of several bacterial clones. The yield is a small amount of impure plasmid DNA, which is sufficient for analysis by restriction digest and for some cloning techniques.

In the latter, much larger volumes of bacterial suspension are grown from which a maxi-prep can be performed. Essentially this is a scaled-up miniprep followed by additional purification. This results in relatively large amounts (several micrograms) of very pure plasmid DNA. In recent times many commercial kits have been created to perform plasmid extraction at various scales, purity and levels of automation. Commercial services can prepare plasmid DNA at quoted prices below $300/mg in milligram quantities and $15/mg in gram quantities (early 2007).

Conformations
Plasmid DNA may appear in one of five conformations, which (for a given size) run at different speeds in a gel during electrophoresis. The conformations are listed below in order of electrophoretic mobility (speed for a given applied voltage) from slowest to fastest:
y y

"Nicked Open-Circular" DNA has one strand cut. "Relaxed Circular" DNA is fully intact with both strands uncut, but has been enzymatically "relaxed" (supercoils removed). You can model this by letting a twisted extension cord unwind and relax and then plugging it into itself. "Linear" DNA has free ends, either because both strands have been cut, or because the DNA was linear in vivo. You can model this with an electrical extension cord that is not plugged into itself. "Supercoiled" (or "Covalently Closed-Circular") DNA is fully intact with both strands uncut, and with a twist built in, resulting in a compact form. You can model this by twisting an extension cord and then plugging it into itself. "Supercoiled Denatured" DNA is like supercoiled DNA, but has unpaired regions that make it slightly less compact; this can result from excessive alkalinity during plasmid preparation.

The rate of migration for small linear fragments is directly proportional to the voltage applied at low voltages. At higher voltages, larger fragments migrate at continually increasing yet different rates. Therefore the resolution of a gel decreases with increased voltage. At a specified, low voltage, the migration rate of small linear DNA fragments is a function of their length. Large linear fragments (over 20kb or so) migrate at a certain fixed rate regardless of length. This is because the molecules 'resperate', with the bulk of the molecule following the leading end through the gel matrix. Restriction digests are frequently used to analyse purified plasmids. These enzymes specifically break the DNA at certain short sequences. The resulting linear fragments form 'bands' after gel electrophoresis. It is possible to purify certain fragments by cutting the bands out of the gel and dissolving the gel to release the DNA fragments. Because of its tight conformation, supercoiled DNA migrates faster through a gel than linear or open-circular DNA.

Bacteriophage

The structure of a typical myovirus bacteriophage

A bacteriophage (from 'bacteria' and Greek phagein "to eat") is any one of a number of viruses that infect bacteria. Bacteriophages are among the most common biological entities on Earth.[1] The term is commonly used in its shortened form, phage. Typically, bacteriophages consist of an outer proteincapsid enclosing genetic material. The genetic material can be ssRNA, dsRNA, ssDNA, or dsDNA ('ss-' or 'ds-' prefix denotes singlestrand or double-strand) along with either circular or linear arrangement. Phages are estimated to be the most widely distributed and diverse entities in the biosphere.[2] Phages are ubiquitous and can be found in all reservoirs populated by bacterial hosts, such as soil or the intestines of animals. One of the densest natural sources for phages and other viruses is sea water, where up to 9108virions per milliliter have been found in microbial mats at the surface,[3] and up to 70% of marine bacteria may be infected by phages.[4] They have been used for over 90 years as an alternative to antibiotics in the former Soviet Union and Eastern Europe as well in

France.[5] They are seen as a possible therapy against multi drug resistant strains of many bacteria.[6]

Replication
Bacteriophages may have a lytic cycle or a lysogenic cycle, and a few viruses are capable of carrying out both. With lytic phages such as the T4 phage, bacterial cells are broken open (lysed) and destroyed after immediate replication of the virion. As soon as the cell is destroyed, the phage progeny can find new hosts to infect. Lytic phages are more suitable for phage therapy. Some lytic phage undergo a phenomenon known as lysis inhibition where completed phage progeny will not immediately lyse out of the cell if there is a high extracellular phage concentrations. This mechanism is not identical to that of temperate phage going dormant and is usually temporary. In contrast, the lysogenic cycle does not result in immediate lysing of the host cell. Those phages able to undergo lysogeny are known as temperate phages. Their viral genome will integrate with host DNA and replicate along with it fairly harmlessly, or may even become established as a plasmid. The virus remains dormant until host conditions deteriorate, perhaps due to depletion of nutrients, then the endogenous phages (known as prophages) become active. At this point they initiate the reproductive cycle, resulting in lysis of the host cell. As the lysogenic cycle allows the host cell to continue to survive and reproduce, the virus is reproduced in all of the cells offspring. Sometimes prophages may provide benefits to the host bacterium while they are dormant by adding new functions to the bacterial genome in a phenomenon called lysogenic conversion. An eminent example is the conversion of a harmless strain of Vibrio cholerae by a phage into a highly virulent one, which causes cholera. This is why temperate phages are not suitable for phage therapy.

Attachment and penetration

An electron micrograph of bacteriophages attached to a bacterial cell. These viruses are the size and shape of coliphage T1 To enter a host cell, bacteriophages attach to specific receptors on the surface of bacteria, including lipopolysaccharides, teichoic acids, proteins, or even flagella. This specificity means that a bacteriophage can only infect certain bacteria bearing receptors to which they can bind, which in turn determines the phage's host range. Host growth conditions also influence the ability of the phage to attach and invade bacteria.[10] As phage virions do not move independently, they must rely on random encounters with the right receptors when in solution (blood, lymphatic circulation, irrigation, soil water, etc.). Myovirus bacteriophages use a hypodermic syringe-like motion to inject their genetic material into the cell. After making contact with the appropriate receptor, the tail fibers bring the base plate closer to the surface of the cell known as reversible binding. Once attached completely irreversible binding is initiated, the tail contracts, possibly with the help of ATP present in the tail,[4] injecting genetic material through the bacterial membrane. Podoviruses lack a elongated tail sheath similar to that of a myovirus so will instead use its small tail fibers in a teeth-like manner to enzymatically degrade a portion of the cell membrane before inserting its genetic material.

Synthesis of proteins and nucleic acid

Within minutes, bacterial ribosomes start translating viral mRNA into protein. For RNA-based phages, RNA replicase is synthesized early in the process. Proteins modify the bacterial RNA polymerase so that it preferentially transcribes viral mRNA. The hosts normal synthesis of proteins and nucleic acids is disrupted, and it is forced to manufacture viral products instead. These products go on to become part of new virions within the cell, helper proteins which help assemble the new virions, or proteins involved in cell lysis. Walter Fiers (University of Ghent, Belgium) was the first to establish the complete nucleotide sequence of a gene (1972) and of the viral genome of Bacteriophage MS2 (1976).[11]

Virion assembly
In the case of the T4 phage, the construction of new virus particles involves the assistance of helper proteins. The base plates are assembled first, with the tails being built upon them afterwards. The head capsids, constructed separately, will spontaneously assemble with the tails. The DNA is packed efficiently within the heads. The whole process takes about 15 minutes.

Diagram of a typical tailed bacteriophage structure

Release of virions
Phages may be released via cell lysis, by extrusion, or, in a few cases, by budding. Lysis, by tailed phages, is achieved by an enzyme called endolysin, which attacks and breaks down the cell wall peptidoglycan. An altogether different phage type, the filamentous phages, make the host cell continually secrete new virus particles. Released virions are described as free, and, unless defective, are capable of infecting a new bacterium. Budding is associated with certain Mycoplasma phages. In contrast to virion release, phages displaying a lysogenic cycle do not kill the host but, rather, become long-term residents as prophage.

Phage therapy
Phages were discovered to be anti-bacterial agents, but the medical trials performed in western countries were sub-standard to the point of not being scientifically viable; this was because the early tests were conducted poorly and without an idea of what a phage was. Phage therapy was shortly thereafter ruled out as untrustworthy much because many of the trials were conducted on totally unrelated diseases such as allergies and viral infections. Antibiotics were discovered some

years later and marketed widely, popular because of their broad spectrum and easier to manufacture in bulk, store, and prescribe. Hence development of phage therapy was largely abandoned in the West, but continued throughout 1940s in the Soviet Union for treating bacterial infections, with widespread use including the soldiers in the Red Armymuch of the literature was published in Russian or Georgian, and unavailable for many years in the West. Their use has continued since the end of the Cold War in Georgia and elsewhere in Eastern Europe. A monograph written by Nina Chanishvili"A Literature Review of the Practical Application of Bacteriophage Research" was published in 2009, in Tbilisi, Georgia.[12] The monograph gives the most thorough analysis of the results of phage therapy according to the data given in the old Soviet scientific literature. The first regulated clinical trial of efficacy in Western Europe (against ear infections caused by Pseudomonas aeruginosa) was reported in the journal Clinical Otolaryngology in August 2009.[13] Meanwhile, Western scientists are developing engineered viruses to overcome antibiotic resistance, and experimenting with tumor-suppressing agents.[citation needed]. One potential treatment currently under development is a phage designed to destroy MRSA. [14]

In the environment
Main article: Marine bacteriophage Metagenomics has allowed the in-water detection of bacteriophages that was not possible previously. These investigations revealed that phage are much more abundant in the water column of both freshwater and marine habitats than previously thought[15] and that they can cause significant mortality of bacterioplankton. Methods in phage community ecology have been developed to assess phage-induced mortality of bacterioplankton and its role for food web process and biogeochemical cycle to genetically fingerprint phage communities or populations and estimate viral biodiversity by metagenomics. The lysis of bacteria by phages releases organic carbon that was previously particulate (cells) into dissolved forms, which makes the carbon more available to other organisms. Phages are not only the most abundant biological entities but probably also the most diverse ones. The majority of the sequence data obtained from phage communities has no equivalent in databases. These data and other detailed analyses indicate that phage-specific genes and ecological traits are much more frequent than previously thought. In order to reveal the meaning of this genetic and ecological versatility, studies have to be performed with communities and at spatiotemporal scales relevant for microorganisms.[2] Bacteriophages have also been used in hydrological tracing and modelling in river systems especially where surface water and groundwater interactions occur. The use of phages is preferred to the more conventional dye marker because they are significantly less absorbed when passing through ground-waters and they are readily detected at very low concentrations.[16]

Role in food fermentation

A broad number of food products, commodity chemicals, and biotechnology products are manufactured industrially by large-scale bacterial fermentation of various organic substrates.

Because enormous amounts of bacteria are being cultivated each day in large fermentation vats, the risk of bacteriophage contamination could rapidly bring fermentation to a halt. The resulting economic setback is a serious threat in these industries. The relationship between bacteriophages and their bacterial hosts is very important in the context of the food fermentation industry. Sources of phage contamination, measures to control their propagation and dissemination, and biotechnological defense strategies developed to restrain phages are of interest. The dairy fermentation industry has openly acknowledged the problem of phages and has been working with academia and starter culture companies to develop defense strategies and systems to curtail the propagation and evolution of phages for decades.[2]

Other areas of use

In August, 2006 the United States Food and Drug Administration (FDA) approved using bacteriophages on cheese to kill the Listeria monocytogenes bacteria, giving them GRAS status (Generally Recognized As Safe).[17] In July 2007, the same bacteriophages were approved for use on all food products.[18] There is on going research to see if lytic phage are a viable option to control E. coli 0157:H7 and other food-borne pathogens in various food products. Government agencies in the West have for several years been looking to Georgia and the Former Soviet Union for help with exploiting phages for counteracting bioweapons and toxins, such as anthrax and botulism.[19] There are many developments with this amongst research groups in the US. Other uses include spray application in horticulture for protecting plants and vegetable produce from decay and the spread of bacterial disease. Other applications for bacteriophages are as a biocide for environmental surfaces, e.g., in hospitals, and as a preventative treatment for catheters and medical devices prior to use in clinical settings. The technology now exists for phages to be applied to dry surfaces, e.g., uniforms, curtains, even sutures for surgery. Clinical trials reported in the Lancet[13] show success in veterinary treatment of pet dogs with otitis. Phage display is a different use of phages involving a library of phages with a variable peptide linked to a surface protein. Each phage's genome encodes the variant of the protein displayed on its surface (hence the name), providing a link between the peptide variant and its encoding gene. Variant phages from the library can be selected through their binding affinity to an immobilized molecule (e.g. Botulism toxin) to neutralize it. The bound selected phages can be multiplied by re-infecting a susceptible bacterial strain, thus allowing them to retrieve the peptides encoded in them for further study. The SEPTIC bacterium sensing and identification method utilizes the ion emission and its dynamics during phage infection and offers high specificity and speed for detection.

Group II intron

Structure of group II intron Group II introns are a large class of self-catalytic ribozymes as well as mobile genetic element found within the genes of all three domains of life. Ribozyme activity (e.g., Self-splicing) can occur under high salt conditions in vitro, however, assistance from proteins is required for in vivo splicing. In contrast to group I introns, intron excision occurs in the absence of GTP and involves the formation of a lariat, with an A-residue branchpoint strongly resembling that found in lariats formed during splicing of nuclear pre-mRNA. It is hypothesized that pre-mRNA splicing (see spliceosome) may have evolved from group II introns due to the similar catalytic mechanism as well the structural similarity of the Domain V substructure to the U6/U2 extended snRNA.

Finally, their ability to site-specifically mobilize to new DNA sites has been exploited as a tool for biotechnology.

Structure and catalytic site

The secondary structure of group II introns is characterized by six typical stem-loop structures, also called domains I to VI or D1 to D6. The domains radiate from a central core that brings the 5' and 3' splice junctions into close proximity. The proximal helix structures of the six domains are connected by a few nucleotides in the central region (linker or joiner sequences). Due to its enormous size, the domain 1 was divided further into subdomains a, b, c, and d. Sequence differences of group II introns were identified which led to a further division into subgroups IIA and IIB. Group II introns also form very complicated RNA Tertiary Structure. Group II introns possess only a very few conserved nucleotides, and the nucleotides important for the catalytic function are spread over the complete intron structure. The few strictly conserved primary sequences are the consensus at the 5' and 3' splicing site (... GUGYG&... and ...AY ...), some of the nucleotides of the central core (joiner sequences), a relatively high number of nucleotides of D5 and some short sequence stretches of D1. The unpaired adenosine in D6 marked by an asterisk (7 or 8 nt away from the 3' splicing site, respectively) is also conserved and plays a central role in the splicing process. In 2005, A. De Lencastre et al. found that during splicing of Group II introns, all reactants are preorganized before the initiation of splicing. The branch site, both exons, the catalytically essential regions of D5 and J2/3, and epsilonepsilon' are in close proximity before the first step of splicing occurs. In addition to the bulge and AGC triad regions of D5, the J2/3 linker region, the epsilonepsilon' nucleotides and the coordination loop in D1 are crucial for the architecture and function of the active-site.

Group II catalytic intron

Group II catalytic introns are found in rRNA, tRNA and mRNA of organelles in fungi, plants and protists, and also in mRNA in bacteria. They are large self-splicing ribozymes and have 6 structural domains (usually designated dI to dVI). This model and alignment represents only domains V and VI. A subset of group II introns also encode essential splicing proteins in intronicORFs. The length of these introns can therefore be up to 3kb. Splicing occurs in almost identical fashion to nuclear pre-mRNA splicing with two transesterification steps. The 2' hydroxyl of a bulged adenosine in domain VI attacks the 5' splice site, followed by nucleophilic attack on the 3' splice site by the 3' OH of the upstream exon. Protein machinery is required for splicing in vivo, and long range intron-intron and intron-exon interactions are important for splice site positioning. Group II introns are further sub-classified into groups IIA and IIB, which differ in splice site consensus, and the distance of the bulged adenosine in domain VI (the prospective branch point forming the lariat) from the 3' splice site.