Literature reviewTransposonsBy John McQuillan |
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Transposons, or Transposable Genetic Elements (TGE), or "Jumping Genes", are lengths of DNA capable of transposition, that is movement, from a plasmid to a chromosome (or another plasmid) and vice-versa in prokaryotes, and from one part of a chromosome to another (or to another chromosome) in eukaryotes. In prokaryotic organisms such as Escherichia coli, it is possible for transposons to be transferred along with plasmid or chromosomal DNA, either independently or as a cointegrate, from one organism to another during conjugation. Some plasmids, such as pMB9, can become mobilised during conjugation, moving along with chromosomal or other plasmid DNA, into a recipient cell, without having instigated the conjugation. A transposon consisting of genes that code not only for its transposition, but also for unrelated activities such as antibiotic and heavy metal resistance is known as a complex transposon (Singleton and Sainsbury, 1993). It is this additional coding that differentiates complex transposons from simple insertion sequences. Insertion sequences (IS) are TGEs consisting only of the genes necessary for transposition of the sequence, using the enzyme transposase and inverted base pair repeats. Inverted repeats have the same DNA sequence at either end with opposite orientations as illustrated below: Many transposons also have inverted repeats at either end, although some have direct repeats where the ends are identical as below: The terminal repeats are important for transposition, acting as recognition points for transposase (see below). Transposable elements were first suspected to exist in the 1940's by geneticist Barbara McClintock while carrying out experiments on maize. It was found that breakages were being caused at a specific site on chromosome 9 due to the insertion of new genetic material (McClintock, 1948; cited by Fedoroff, 1985). In prokaryotes, transposons were not discovered until the 1960's when mutant Escherichia coli were found that where unable to ferment galactose (Gal-). These E. coli were found to have DNA (insertion sequences) unrelated to the galactose gene inserted within the gene, causing its inactivation. At the same time study was being undertaken into the spread of bacterial antibiotic resistance. Sections of DNA were found with the ability to move within the genome, and between related bacterial species, that carried genes conferring antibiotic resistance. It was originally thought that these genes were rare and carried on plasmids, and it was not until 1974 that actual antibiotic resistance transposons were discovered (Berg, C.M. and Berg, D.E. 1984; Hedges and Jacob, 1974, cited by Heffron, 1983). The continued and heavy use of antibiotics in agriculture and medicine has resulted in the selective culture of pathogenic bacteria resistant to the most popular antibiotics (Simonsen and Levin, 1988), such as penicillin, tetracycline, kanamycin and ampicillin. Transposons can carry resistance to the antibiotics used to treat many diseases, from the annoying such as diarrhoea to the more serious such as gonorrhoea to the potentially fatal such as pneumonia, infant diarrhoea and tuberculosis. Resistance can be carried on the transposon itself, such as ampicillin resistance (Apr) on Tn3, or on plasmids to which the transposon is attached such as pMB9::Tn103 (plasmid/transposon complex), where resistance to tetracycline (Tcr ) is carried on the pMB9 plasmid. Tn103 (a member of the Tn3 group) is able to instigate cointegrate formation but is not able to independently resolve the cointegrate once formed, due to the inactivity of the tnpR (resolvase) gene. Tn103 also has no antibiotic resistance because the bla (-lactamase) gene is also inactive (Hettle, 1995). The presence of a transposon on a plasmid allows it to become incorporated into the bacterial chromosome or to join with another plasmid. In cases where the transposon is on a plasmid with a conjugative factor, the transposon may then be passed on to a bacterium of the same or similar species, thereby passing on the resistance marker to another bacterium. Types of prokaryotic transposon Prokaryotic transposons fall into three groups: Class I: These are mainly short insertion sequences, and carry only the genes required for the production of the enzymes responsible for their transposition. Class I elements demonstrate both basic, direct, transposition (that is transposition directly from one molecule to another without the formation of any intermediate structure, i.e. a cointegrate) and cointegrate formation. In general, direct transposition is more common, type and frequency depending on the TGE. With Tn9 direct transposition is five times more likely to occur than cointegrate formation, while Tn903 seems to use both methods with equal frequency (Kleckner, 1981). During insertion, from 3 or 4 to 9 base pair repeats are made on the ends of the recipient DNA molecule, that is a short DNA sequence produced immediately before the production of DNA on the inserted transposon, joining the target DNA to the transposon. Class I elements are further subdivided into two groups: Classes IA and IB (see fig. 3 below for layout). Class IA elements are the simplest, being simple insertion sequences, having only the genes for transposition and transposition regulation, i.e. IS1; IS2; etc.. Class IB are composite transposons, also containing genes coding for antibiotic resistance and other products, e.g. Tn681, genes for an enterotoxin; Tn2350, genes for kanamycin resistance (kanr); Tn2901, genes for arginine biosynthesis; etc. (Kleckner, 1981).  Class II: The Class II elements are typified by Tn3. Class II transposons all bear a strong resemblance to one another and to the archetypal Tn3. Transposition is via cointegrate formation followed by resolution. In all Class II elements insertion generates 5 base pair repeats at the ends of the insertion point in the target DNA (see Transposon Tn3 (and relatives) below). Class III: Class III elements are bacteriophages Mu and D108, these bacteriophages share 90% homology in their genetic make-up (Singleton and Sainsbury, 1993). Mu (short for Mutator) is capable of causing mutations in host cells due to its ability to insert randomly in any part of the bacterial genome. Random insertion means that it can insert into a key part of a gene causing its inactivation. Unlike Class I and II elements, there are no terminal inverted repeats in Mu. There are non-symmetrical homologies between the ends and other regions within the bacteriophage, e.g. between one end of the bacteriophage and a region approximately 80 bp in from the opposite end, and a sequence of 7-9 bp in the vicinity of one end which occurs again five times near the opposite end (Kamp and Kahmann, 1981 and 1979, cited in Kleckner, 1981). Replicative transposition is a compulsory part of the life cycle of Mu and D108 whether the life cycle is lysogenic or lytic (Kleckner, 1981; Bétermier et al, 1992). Transposon Tn3 (and relatives) Tn3 is a 4957 base pair class II transposon. At each end it has identical 38 base pair inverted repeats. The transposon also contains the genes for transposase (tnp A), resolvase (tnp R), and -lactamase (bla).  Figure 4 above illustrates the general layout of Tn3, the area marked IRS is the internal resolution site, without this site the resolution of the cointegrate can not take place. IR-L and IR-R are inverted repeats - left and right respectively. The -lactamase is responsible for the transposons ability to confer antibiotic resistance on the bacteria in which it is present. -lactamase is able to hydrolyse the -lactam ring present on -lactam antibiotics such as penicillins. The hydrolysis of the ring renders the antibiotic innocuous to the organism, allowing growth and reproduction. Tn3 provides resistance to ampicillin (Ap). Ampicillin can therefore be used in growth media to screen for the presence of Tn3 in a bacterial colony (Heffron et al, 1983; Kretschmer and Cohen, 1979; Heffron et al, 1979).
Any defect or deletion in the IRS or the tnpR gene can prevent resolution from taking place, locking the plasmid/transposon complex to the chromosome/plasmid with which it is cointegrated. If the internal resolution site is intact but the tnpR gene is defective, resolution can still occur through the action of resolvase from another transposon (present on another plasmid) or by the action of the host cells recA system. tnpA is the gene responsible for transposase, this is vital for cointegrate formation, repression of its expression prevents transposition. tnpR is the gene which codes for resolvase, this divides the cointegrate into its two original molecules, the donor plasmid on which the transposon was initially found and the chromosome/plasmid to which a copy of the transposon has now been added. Resolvase is therefore responsible for the recombination of the transposon at the internal resolution site. This is called site specific recombination, requiring short, specific sequences of double stranded DNA (dsDNA) without the synthesis or breakdown of any DNA (Singleton and Sainsbury, 1993). Resolvase also acts as an inhibitor of both transposase and resolvase production, therefore acting as an inhibitor of transposition, possibly by binding to both the tnpA and tnpR promoter sites, preventing the expression of both genes (Kitts et al, 1982; Symonds 1988; Grindley and Reed, 1985; Heffron, 1983). Tn3 also exhibits transposition immunity, that is if a molecule has a Tn3 transposon already present, the chances that another will be allowed to insert are greatly reduced with transposition frequencies less than 5% that of a non-immune molecule, i.e. a molecule without a transposon (Grindley and Reed, 1985). The immunity appears to be a product of the resolvase, and its recognition of a terminal inverted repeat on a target molecule. Transposase from any source within a cell finding a terminal inverted repeat on a potential target molecule will inhibit a further transposition event from occurring. Where a third inverted repeat is present on a molecule, the likelihood of intramolecular movement of Tn3 is reduced. Transposition immunity is cis acting, i.e. the presence of a Tn3 terminal inverted repeat on one molecule does not affect the frequency of transposition on other molecules (Grindley and Reed, 1985; Kleckner, 1981). There are around 20 members of the Tn3 group including Tn1, Tn2, Tn4 and Tn501. Some of these transposons code for different antibiotic resistance, e.g. Tn4 codes for resistance to ampicillin, streptomycin and sulphonamides, while Tn501 codes for resistance to mercury (Kleckner, 1981; Singleton and Sainsbury, 1993). Mechanisms of transposition in prokaryotes As mentioned earlier, transposition can occur directly, by transfer of a transposable element from one plasmid to another plasmid or chromosome with no intermediate, or, via the formation of a cointegrate followed by resolution. The latter always yields two transposons on separate plasmids or plasmid/chromosome. Transposition is a rare event, depending on the physicochemical conditions within the cell and the element involved, in general occurring at around 10-4 - 10-7 times per element per generation (Kleckner, 1981). Concentrating on Tn3, cointegrate formation requires the action of the enzyme transposase, encoded by the tnpA gene, and resolution requires resolvase, encoded by the tnpR gene, and also the presence on the transposon of a resolution site and intact terminal inverted repeats. These sites on Tn3 are illustrated on figure 4 above. The terminal inverted repeats and the resolution site must be present in cis for a full transposition event to occur (Heffron, 1983; Heffron et al, 1979). The resolvase and the transposase can, if necessary, be provided by an intact Tn3 elsewhere in the genome, but with out the correct terminal repeat sequences and internal resolution site they will be unable to carry out their function. The terminals and the resolution site act as recognition sites and points of reference for the enzymes and are involved in binding the enzymes to the transposon, this is particularly true of the resolution site/resolvase complex (Heffron, 1983; Heffron et al 1979). The mechanics of the operation for Tn3 (and other Class II elements) can be broken down into the two steps: 1) cointegrate formation and 2) resolution of the cointegrate. Step 1 requires the action of transposase, the presence of intact terminal inverted repeats, and suitable insertion sites on a target DNA molecule, preferably without another intact Tn3. Tn3 does not show great insertion site specificity but seems to prefer "hot-spots" were insertion is more likely. Regions of high A and T concentration seem to be preferred for insertion although preference also seems to depend on other factors such as the DNA secondary structure (Davies and Hutchison, 1995; Heffron, 1983). Step 2 requires the action of resolvase and an intact internal resolution site.  The process of transposition is still not fully understood. For Tn3, transposition is believed to begin when transposase cleaves the target DNA at the insertion site (see fig. 6). The synthesis of five bp repeats by DNA Polymerase I, joins the ends of the target molecule to the transposon and new DNA is synthesised along the lengths of the two strands of transposon DNA. Eventually, when replication is complete, there are two identical copies of the transposon which link the donor and recipient molecules as a cointegrate (see figures 7 & 8 below).  Figure 7 above illustrates two possible routes for transposition. Route 1 is the obligatory pathway taken by Class II elements (e.g. Tn3). This pathway includes cointegrate formation followed by resolution generating two copies of the transposon on two separate molecules. Route ‚ illustrates the direct insertion of a transposon from a donor into a recipient molecule, resulting in the breakdown of the donor DNA. Both of these routes are symmetrical, that is the DNA is being synthesised via replication forks from both ends of the transposon. In figure 8 (below), the detail of replication is more clearly illustrated with the replication forks clearly shown. The full cointegrate is also illustrated in both isometric and topological view to show the relationship of the two transposons to the overall conformation of the cointegrate.  Resolution involves the recombination of the DNA making up the two transposons at the internal resolution site. The IRS consists of three internal sites: I, II and III. Sites II and III are used for binding the resolvase and site I is the point of cleavage. Within the IRS are also sited the promoter regions for both tnpA (within the region of site I) and tnpR (between the regions of sites I and II) genes. The transposons are cleaved and recombined in the manner illustrated at the end of route 1 in figure 7, leaving both strands with a combination of both original and new DNA, joined at the res site (Grindley and Reed, 1985; Kleckner, 1981). An asymmetric model has also been proposed in which DNA synthesis is
carried out only at one end of the transposon. Instead of cutting strands
at both ends, the transposase cuts recipient and donor strands at one end
only and a replication fork moves from one end to the other. In this model
both cointegrates and simple insertions can be carried out as per the symmetrical
model, resolution would be unaffected (Grindley and Reed, 1985; Kleckner,
1981).
Effects of temperature on transposition The rate of transposition of prokaryotic transposons has been shown to be temperature dependent (Kretschmer and Cohen, 1979; Cornelis, 1979). 
Figure 9 and table 1 above (adapted from Kretschmer and Cohen, 1979) both show the relationship between temperature and transposition. Transposon Tn3 in Escherichia coli has an optimum between 26oC and 30oC, above 30oC the rate falls of rapidly with a direct negative linear relationship between frequency and temperature, as illustrated by the graph. These results are similar to results obtained for Tn951 from two different plasmid sources in two different organisms detailed in table 2 below (from Cornelis, 1979). The different organisms were used to show that the rate of transposition was affected by temperature and not by the growth rate of the organisms (Yersinia enterocolitica has a faster growth rate at 30oC than at 37oC). All of these results indicate that one or more of the processes involved in transposition are affected by temperature.
Transposition events depend on the ability of transposase and resolvase to carry out their functions as described above (Mechanisms of transposition in prokaryotes). Changes in the physiological conditions in the cell may cause a conformational change in either the transposase or the resolvase, making them less efficient. In the case of Tn951 it is suggested by Cornelis, 1979, that it is the action of cointegrate formation that is influenced by the temperature variation. In eukaryotes, transposons are generally more complex than in prokaryotes. In maize, the plant in which transposons were originally discovered (see Discovery above), the two TGEs, Ac and Ds, show a strong similarity to prokaryotic transposons with terminal IRs and a gene coding for a transposase. The transposase gene, in common with other eukaryotic genes, codes for a primary mRNA that contains introns, these have to be removed by further processing. The Ac element is a fully functional transposon capable of movement from one part of the genome to another, while Ds elements are unable to move of their own accord, due to deletions in the transposase gene (Watson et al, 1992). In maize, Ac elements cause mutations by interrupting genes, these mutations are reversible if the element moves on. The Ds element does not give rise to reversion mutation unless there is a functioning Ac element present, producing transposase, this is the same as the trans action of the tnpA and tnpR genes in bacterial transposons (Watson et al, 1992). In Drosophila, P elements, like the Ac element in maize and the bacterial transposons, consists of a transposase gene and terminal IRs. Most P elements suffer from deletions which prevents their transposition without an external source of transposase. P elements are only active in germ line cells. The production of active transposase is dependent on the ability of the cell to splice the exons on the primary mRNA, this is only possible in germ line cells. In somatic cells, the splicing of the final exon to the preceding one, does not occur, this results in a non functioning transposase that may actively inhibit transposition (Watson et al, 1992). Yeast contains transposons called Ty elements. Ty elements are arranged differently from bacterial transposons, having long direct terminal repeats of around 330 bp called sequences (Watson et al, 1992; Singleton and Sainsbury, 1993). The direct repeats carry promoter regions for RNA polymerase. Transposition occurs through an RNA intermediate and involves a reverse transcriptase coded for by a gene on the Ty element. This type of transposon, using RNA and reverse transcriptase is called a retrotransposon (Watson et al, 1992; Singleton and Sainsbury, 1993). Ty elements can cause mutation in the same way as all other transposons, but, unlike other transposons, Ty elements can also activate some genes. Activation is caused by the strong influence of the RNA polymerase promoter regions in the terminal repeats when they are inserted into a suitable region in the vicinity of a susceptible gene, transcription of that gene occurs along with the transcription of the Ty element genes (Watson et al, 1992; Singleton and Sainsbury, 1993). Mammal genomes also contain transposons, LINEs (Long Interspersed Nuclear Sequences), SINEs (Short Interspersed Nuclear Sequences) and endogenous retroviruses. It is believed that they show a system of transposition similar to that of the Ty elements in yeast, that is, retrotransposition (Gabriel, A. 1995). In humans, the genome may contain 50,000 - 100,000 copies of a particular LINE sequence called L 1 Hs (LINE 1 from Homo sapiens). LINEs range from 6-7kb in length and consist of two variable length target site duplications at either end. Within the LINE there are two open reading frames (reading frames without stop codons) and a 3'-poly (A) tail. The mechanism of transposition used by LINEs is not known, they are believed to be retrotransposons with a full length RNA intermediate. The RNA intermediate codes for reverse transcriptase and other enzymes, and acts as a template for the production of new DNA and its insertion into the genome (Gabriel, A. 1995). SINEs exist in greater numbers than LINEs, and are found within the human genome every 5 to 10kb, normally in introns and other regions of genes that normally remain untranslated. The most common SINEs in the human genome are the Alu elements, called after the presence within the element of a restriction site for Alu I. Alu elements are 100 to 500 bases long with variable length target site duplications at each end, they have 3'-poly (A) tails and a Alu I restriction site. Unlike LINE elements, SINEs are not able to code for reverse transcriptase, relying therefore on an external source. The likeliest sources being LINEs and endogenous retroviruses (Gabriel, A. 1995). Endogenous retroviruses are believed to have arisen from the insertion of retroviral proviruses (DNA produced from retroviral RNA and inserted into the host genome) into the genome of germ cells and then transmitted vertically. Endogenous retroviruses are therefore found in every cell of an organism. In humans there are a number of different classes of endogenous retrovirus called HERVs (human endogenous retroviruses). Endogenous retroviruses are 5 to 9kb in length and have fixed length target site duplications at each end. Also at each end there are long terminal repeat sequences. They carry genes coding for the enzymes necessary for transposition. The majority of HERVs have a strong resemblance to exogenous retroviruses and to endogenous retroviruses in other species. The similarity and position in the genome of certain endogenous retroviruses in primates indicates that the retroviruses from which they are derived were inserted into archetypal primate germ line cells early in their evolution (Gabriel, A. 1995). It has been conjectured that transposons were responsible for the burst of new organisms that appeared during the Cambrian period of the Earth's prehistory (Travis, 1992). It is possible that transposable elements, particularly those able to change the regulation of genes responsible for development, may have caused bursts of speciation. This differentiation may have led to the development of new species, some with survival advantages over their progenitors and unaffected relatives. In humans, a TGE has been found which has inserted into the factor VIII gene on the X chromosome, producing haemophilia A (Dombroski et al, 1991). The medfly, a pest in fruit growing areas, is controlled by sterilisation through irradiation of embryos, unfortunately both male and female embryos are sterilised, and as the flies only mate once, this reduces the effectiveness of the procedure as sterile males can mate with sterile females. A transposon from the fruit fly Drosophila hydei, called the Minos element has been used to carry genes for white eye into the medfly. It is hoped that the white eye gene will be able to be inserted along with genes which will cause the fly embryos to develop as males with white eyes, making selection for release easier (Savakis et al, reported in New Scientist, 1996). Transposons are important to both the medical profession and to the biotechnology industry. Medicinally their importance lies not only in their ability to confer antibiotic resistance to medically important bacteria, but also in their potential for future treatments using gene therapy. In the biotechnology industries, transposons have the potential to provide a vector for the insertion of new genetic material into the genome of cells in which it would not normally be found. In the case of gene therapy, the position in the gene in which the transposon inserts will have to be carefully selected to prevent any adverse effects on neighbouring genes. Also, the potential effect on the environment of genetically modified organisms will have to be carefully assessed before any release. Transposons have a continuing role in the biotechnology and pharmaceutical industries offering a wide range of possibilities for genetic modification. Considering the above possibilities and the importance of transposons in many different prokaryotic and eukaryotic cells, they are worthy of continued study.
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