The Tn3 transposon is a 4957 base pair mobile genetic element, found in prokaryotes. It encodes three proteins:
Initially discovered as a repressor of transposase, resolvase also plays a role in facilitating Tn3 replication (Sherratt 1989).
The transposon is flanked by a pair of 38bp inverted repeats.
This first stage is catalysed by transposase.
The plasmid containing the transposon (the donor plasmid) fuses with a host plasmid (the target plasmid). In the process, the transposon and a short section of host DNA are replicated. The end product is a 'cointegrate' plasmid containing two copies of the transposon.
Shapiro (1978)proposed the following mechanism for this process:
The diagrams on the right illustrate the way in which the positions of the cleavages lead to the replication of certain regions once the plasmids have fused.
To separate the host and target molecules Tn3 resolvase executes site-specific recombination between the old and new copy of transposon at a specific site called res, which is present in each copy of the transposon. Res is 114 bp long and it consists of 3 sub-sites, namely sites I, II and III. Each of these sites is of different lengths (28, 34 and 25bp, respectively) and they are unevenly spaced with 22bp separating sites I and II and only 5bp between sites II and III. The sites consist of 6bp inverted repeat motifs flanking a central sequence of variable length. These motifs act as binding sites for resolvase, so that each site binds a resolvase dimer but with varying affinity and probably a slightly different protein-DNA complex architecture.All three sub-sites are essential for recombination.
At recombination, two directly repeated res sites with resolvase dimers bound to each sub-site, come together to form a large complex structure called the synaptosome. Resolvase bound to sites II and III initiates the assembly of this complex. In this structure, exact architecture of which is still unclear, two res sites are intertwined in such a way as to juxtapose two copies of site I, allowing resolvase dimers bound to each site to form a tetramer. Again, it is the interaction between the resolvase dimers bound at accessory sites (sites II and III) and resolvase at site I that causes the two dimers to synapse and form a tetramer. After the tetramer is formed it becomes activated and the top and bottom DNA strands are simultaneously cleaved in the middle of the site I with a 2bp overhang. The strand exchange ensues by as yet unknown mechanism with a resulting net rotation of 180°. The strand exchange is then followed by the religation (Stark et al., 1992). Recombination between two directly repeated res sites separates, or resolves, the "cointegrate" into two original molecules, each one now containing a copy of the Tn3 transposon. After resolution these two molecules remain linked as a simple two-noded catenane which can be easily separated in vivo by a type II topoisomerase (Grindley 2002). Wild type resolvase system absolutely requires a supercoiled substrate and that the recombination sites are oriented in a direct repeat on the same DNA molecule. However, a number of "deregulated" or "hyperactive" mutants that have lost the requirement for the accessory sites have been isolated. These mutants are capable of catalysing recombination between two copies of site I only, which basically reduces the recombination site size from 114bp to only 28bp.Furthermore, these mutants have no supercoiling or connectivity requirements (Arnold et al., 1999) and have been shown to work in mammalian cells. Hyperactive resolvase mutants have so far proven useful in creating resolvases with altered sequence specificity but also in structural work.
The entire resolvase recombination reaction can be reproduced in vitro, requiring only resolvase, a substrate DNA and multivalent cations, using either wild type protein or hyperactive mutants.
Hyperactive resolvase mutants, if further developed, could become an alternative to Cre and FLP, the most commonly used recombination systems in molecular biology to date.
RuvABC is a complex of three proteins that mediate branch migration and resolve the Holliday junction created during homologous recombination in bacteria. As such, RuvABC is critical to bacterial DNA repair.
Transposase is an enzyme that binds to the end of a transposon and catalyses its movement to another part of the genome by a cut and paste mechanism or a replicative transposition mechanism. The word "transposase" was first coined by the individuals who cloned the enzyme required for transposition of the Tn3 transposon. The existence of transposons was postulated in the late 1940s by Barbara McClintock, who was studying the inheritance of maize, but the actual molecular basis for transposition was described by later groups. McClintock discovered that pieces of the chromosomes changed their position, jumping from one chromosome to another. The repositioning of these transposons allowed other genes for pigment to be expressed. Transposition in maize causes changes in color; however, in other organisms, such as bacteria, it can cause antibiotic resistance. Transposition is also important in creating genetic diversity within species and adaptability to changing living conditions. During the course of human evolution, as much as 40% of the human genome has moved around via methods such as transposition of transposons.
P elements are transposable elements that were discovered in Drosophila as the causative agents of genetic traits called hybrid dysgenesis. The transposon is responsible for the P trait of the P element and it is found only in wild flies. They are also found in many other eukaryotes.
Insertion element is a short DNA sequence that acts as a simple transposable element. Insertion sequences have two major characteristics: they are small relative to other transposable elements and only code for proteins implicated in the transposition activity. These proteins are usually the transposase which catalyses the enzymatic reaction allowing the IS to move, and also one regulatory protein which either stimulates or inhibits the transposition activity. The coding region in an insertion sequence is usually flanked by inverted repeats. For example, the well-known IS911 is flanked by two 36bp inverted repeat extremities and the coding region has two genes partially overlapping orfA and orfAB, coding the transposase (OrfAB) and a regulatory protein (OrfA). A particular insertion sequence may be named according to the form ISn, where n is a number ; this is not the only naming scheme used, however. Although insertion sequences are usually discussed in the context of prokaryotic genomes, certain eukaryotic DNA sequences belonging to the family of Tc1/mariner transposable elements may be considered to be, insertion sequences.
Tn10 is a transposable element, which is a sequence of DNA that is capable of mediating its own movement from one position in the DNA of the host organism to another. There are a number of different transposition mechanisms in nature, but Tn10 uses the non-replicative cut-and-paste mechanism. The transposase protein recognizes the ends of the element and cuts it from the original locus. The protein-DNA complex then diffuses away from the donor site until random collisions brings it in contact with a new target site, where it is integrated. To accomplish this reaction the 50 kDa transposase protein must break four DNA strands to free the transposon from the donor site, and perform two strand exchange reactions to integrate the element at the target site. This leaves two strands unjoined at the target site, but the host DNA repair proteins take care of this. The target site selection is essentially random, but there is a preference for the sequence 5'-GCTNAGC-3'. The 6-9 base pairs that flank the sequence also influence selection of the insertion site.
Cre-Lox recombination is a site-specific recombinase technology, used to carry out deletions, insertions, translocations and inversions at specific sites in the DNA of cells. It allows the DNA modification to be targeted to a specific cell type or be triggered by a specific external stimulus. It is implemented both in eukaryotic and prokaryotic systems. The Cre-lox recombination system has been particularly useful to help neuroscientists to study the brain in which complex cell types and neural circuits come together to generate cognition and behaviors. NIH Blueprint for Neuroscience Research has created several hundreds of Cre driver mouse lines which are currently used by the worldwide neuroscience community.
Cre recombinase is a tyrosine recombinase enzyme derived from the P1 bacteriophage. The enzyme uses a topoisomerase I-like mechanism to carry out site specific recombination events. The enzyme (38kDa) is a member of the integrase family of site specific recombinase and it is known to catalyse the site specific recombination event between two DNA recognition sites. This 34 base pair (bp) loxP recognition site consists of two 13 bp palindromic sequences which flank an 8bp spacer region. The products of Cre-mediated recombination at loxP sites are dependent upon the location and relative orientation of the loxP sites. Two separate DNA species both containing loxP sites can undergo fusion as the result of Cre mediated recombination. DNA sequences found between two loxP sites are said to be "floxed". In this case the products of Cre mediated recombination depends upon the orientation of the loxP sites. DNA found between two loxP sites oriented in the same direction will be excised as a circular loop of DNA whilst intervening DNA between two loxP sites that are opposingly orientated will be inverted. The enzyme requires no additional cofactors or accessory proteins for its function.
Hin recombinase is a 21kD protein composed of 198 amino acids that is found in the bacteria Salmonella. Hin belongs to the serine recombinase family (B2) of DNA invertases in which it relies on the active site serine to initiate DNA cleavage and recombination. The related protein, gamma-delta resolvase shares high similarity to Hin, of which much structural work has been done, including structures bound to DNA and reaction intermediates. Hin functions to invert a 900 base pair (bp) DNA segment within the salmonella genome that contains a promoter for downstream flagellar genes, fljA and fljB. Inversion of the intervening DNA alternates the direction of the promoter and thereby alternates expression of the flagellar genes. This is advantageous to the bacterium as a means of escape from the host immune response.
P1 is a temperate bacteriophage that infects Escherichia coli and some other bacteria. When undergoing a lysogenic cycle the phage genome exists as a plasmid in the bacterium unlike other phages that integrate into the host DNA. P1 has an icosahedral head containing the DNA attached to a contractile tail with six tail fibers. The P1 phage has gained research interest because it can be used to transfer DNA from one bacterial cell to another in a process known as transduction. As it replicates during its lytic cycle it captures fragments of the host chromosome. If the resulting viral particles are used to infect a different host the captured DNA fragments can be integrated into the new host's genome. This method of in vivo genetic engineering was widely used for many years and is still used today, though to a lesser extent. P1 can also be used to create the P1-derived artificial chromosome cloning vector which can carry relatively large fragments of DNA. P1 encodes a site-specific recombinase, Cre, that is widely used to carry out cell-specific or time-specific DNA recombination by flanking the target DNA with loxP sites.
Site-specific recombination, also known as conservative site-specific recombination, is a type of genetic recombination in which DNA strand exchange takes place between segments possessing at least a certain degree of sequence homology. Enzymes known as site-specific recombinases (SSRs) perform rearrangements of DNA segments by recognizing and binding to short, specific DNA sequences (sites), at which they cleave the DNA backbone, exchange the two DNA helices involved, and rejoin the DNA strands. In some cases the presence of a recombinase enzyme and the recombination sites is sufficient for the reaction to proceed; in other systems a number of accessory proteins and/or accessory sites are required. Many different genome modification strategies, among these recombinase-mediated cassette exchange (RMCE), an advanced approach for the targeted introduction of transcription units into predetermined genomic loci, rely on SSRs.
Histone-lysine N-methyltransferase SETMAR is an enzyme that in humans is encoded by the SETMAR gene.
Transposon mutagenesis, or transposition mutagenesis, is a biological process that allows genes to be transferred to a host organism's chromosome, interrupting or modifying the function of an extant gene on the chromosome and causing mutation. Transposon mutagenesis is much more effective than chemical mutagenesis, with a higher mutation frequency and a lower chance of killing the organism. Other advantages include being able to induce single hit mutations, being able to incorporate selectable markers in strain construction, and being able to recover genes after mutagenesis. Disadvantages include the low frequency of transposition in living systems, and the inaccuracy of most transposition systems.
A knockout rat is a genetically engineered rat with a single gene turned off through a targeted mutation used for academic and pharmaceutical research. Knockout rats can mimic human diseases and are important tools for studying gene function and for drug discovery and development. The production of knockout rats was not economically or technically feasible until 2008.
Helitrons are one of the three groups of eukaryotic class 2 transposable elements (TEs) so far described. They are the eukaryotic rolling-circle transposable elements which are hypothesized to transpose by a rolling circle replication mechanism via a single-stranded DNA intermediate. They were first discovered in plants and in the nematode Caenorhabditis elegans, and now they have been identified in a diverse range of species, from protists to mammals. Helitrons make up a substantial fraction of many genomes where non-autonomous elements frequently outnumber the putative autonomous partner. Helitrons seem to have a major role in the evolution of host genomes. They frequently capture diverse host genes, some of which can evolve into novel host genes or become essential for Helitron transposition.
Transposons are semi-parasitic DNA sequences which can replicate and spread through the host's genome. They can be harnessed as a genetic tool for analysis of gene and protein function. The use of transposons is well-developed in Drosophila and in Thale cress and bacteria such as Escherichia coli.
The Sleeping Beauty transposon system is a synthetic DNA transposon designed to introduce precisely defined DNA sequences into the chromosomes of vertebrate animals for the purposes of introducing new traits and to discover new genes and their functions. It is a Tc1/mariner-type system, with the transposase resurrected from multiple inactive fish sequences.
The PiggyBac (PB) transposon is a mobile genetic element that efficiently transposes between vectors and chromosomes via a "cut and paste" mechanism. During transposition, the PB transposase recognizes transposon-specific inverted terminal repeat sequences (ITRs) located on both ends of the transposon vector and efficiently moves the contents from the original sites and integrates them into TTAA chromosomal sites. The powerful activity of the PiggyBac transposon system enables genes of interest between the two ITRs in the PB vector to be easily mobilized into target genomes. The TTAA-specific transposon piggyBac is rapidly becoming a highly useful transposon for genetic engineering of a wide variety of species, particularly insects. They were discovered in 1989 by Malcolm Fraser at the University of Notre Dame.
Transposition is the process by which a specific genetic sequence, known as a transposon, is moved from one location of the genome to another. Simple, or conservative transposition, is a non-replicative mode of transposition. That is, in conservative transposition the transposon is completely removed from the genome and reintegrated into a new, non-homologous locus, the same genetic sequence is conserved throughout the entire process. The site in which the transposon is reintegrated into the genome is called the target site. A target site can be in the same chromosome as the transposon or within a different chromosome. Conservative transposition uses the "cut-and-paste" mechanism driven by the catalytic activity of the enzyme transposase. Transposase acts like DNA scissors; it is an enzyme that cuts through double-stranded DNA to remove the transposon, then transfers and pastes it into a target site.
DNA transposons are DNA sequences, sometimes referred to "jumping genes", that can move and integrate to different locations within the genome. They are class II transposable elements (TEs) that move through a DNA intermediate, as opposed to class I TEs, retrotransposons, that move through an RNA intermediate. DNA transposons can move in the DNA of an organism via a single-or double-stranded DNA intermediate. DNA transposons have been found in both prokaryotic and eukaryotic organisms. They can make up a significant portion of an organism's genome, particularly in eukaryotes. In prokaryotes, TE's can facilitate the horizontal transfer of antibiotic resistance or other genes associated with virulence. After replicating and propagating in a host, all transposon copies become inactivated and are lost unless the transposon passes to a genome by starting a new life cycle with horizontal transfer. It is important to note that DNA transposons do not randomly insert themselves into the genome, but rather show preference for specific sites.
Tc1/mariner is a class and superfamily of interspersed repeats DNA transposons. The elements of this class are found in all animals, including humans. They can also be found in protists and bacteria.