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Transposons – the Useful Genetic Tools

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Transposons – the Useful Genetic Tools Biologia, Bratislava, 59/3: 309—318, 2004 REVIEW Transposons – the useful genetic tools Miriam Vizváryová1 &DankaValková2* 1St. Elizabeth Cancer Institute, Heydukova 10,SK-81250 Bratislava, Slovakia 2Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, Mlynská dolina B-2,SK-84215 Bratislava, Slovakia; phone: ++ 421 2 60296509, e-mail:[email protected] VIZVÁRYOVÁ,M.&VALKOVÁ, D., Transposons – the useful genetic tools. Biologia, Bratislava, 59: 309—318, 2004; ISSN 0006-3088. (Biologia). ISSN 1335-6399 (Biologia. Section Cellular and Molecular Biology). Mobile DNA elements, originally discovered in maize more than fifty years ago, have become the indispensable tools for bacterial genetics: so many dif- ferent types of specialised transposon derivatives were constructed so far. The main aim of this article is to summarise the major types of transposon constructs and their application in molecular genetic techniques. Classical in vivo transposition applications include insertional mutagenesis, gene fu- sion and mapping techniques as well as DNA sequencing strategies. Recent applications have extended transposition-based techniques to the analysis of genomes, proteins, protein-DNA complexes and proteomes. Key words: transposon, transposition, in vivo cloning, functional genomics. Abbreviations: ATC, altered target specificity; DR, direct repeat; IR, inverted repeat; IS, insertion sequence; oriT, origin of transfer; Tn, transposon; Tnp, transposase protein; Apr,Kmr,Tmpr,Strr,Cmr,Tcr,Err,resistancetofol- lowing antibiotics: Ampicillin, Kanamycin, Trimetoprim, Streptomycin, Chlo- ramphenicol, Tetracycline, Erytromycin. General features of transposons netic origins (SHAPIRO et al., 1977). The simple insertions, in contrast, are characterised by spe- Transposable elements are discrete DNA segments cific structural features: each insertion contains ex- that can be repeatedly inserted into several sites actly the same set of non permuted transposition in genome. This process is independent of pre- sequences; they are accompanied by duplication of viously recognised mechanisms for the integra- a short target DNA sequence; the ends of trans- tion of DNA molecules and occurs without need posons are terminated by short inverted repeats of DNA sequence homology. The extensive stud- (CALOS &MILLER, 1980). ies – identification and characterisation of mo- KLECKNER (1981) has divided the transpos- bile elements in bacteria started in the late 1960s able elements into three distinct classes, based on and early 1970s. Transposons revealed as the ele- the structural properties, mechanism of transposi- ments diverse in size, structure, specificity of inser- tion and DNA sequence homology: tion, mechanism of transposition, and regulation Class I – the insertion sequence (IS) mod- of movement and might possess several phyloge- ules and composite elements formed from them. * Corresponding author 309 IS modules are short elements, less than 2 kb in the linearised vector-donor is destroyed (BERG et size, encoding only determinants relevant to their al., 1984). The replicative transposition, known own transposition (IS1–IS5,IS102, ISR1). Two for example in Tn1000 (from F factor), is realized copies of certain ISs flanking a DNA segment were by co-integrates that consist of vector and target termed the composite transposons. DNAs (WEINERT et al., 1984). The co-integrates Class II – the transposon (Tn) family, sized canberesolvedinthesecondrecombinationstep. more than 5 kb, containing 38–40 bp inverted While the intermolecular transposition yields in repeats at their ends, which generate 5 bp re- the simple insertion and co-integrates, the in- peats of target DNA during insertion. These usu- tramolecular transposition usually result in dele- ally encode, in addition to transposition functions, tions and/or inversions. the accessory determinants, such as antibiotic and Many of the acquired antibiotic resistance heavy metal resistance. genes, found in enterobacteria and pseudomon- Class III presents the transposing bacterio- ades, are the part of small mobile elements known phages, such as Mu or its derivatives. These pos- as gene cassettes, but similarly other virulence re- sess the genes and sites for transposition as well as lated genes are likely to be found in these cas- the genes for DNA replication, phage development settes. Together they form integrons, the mobile and cell lysis. elements often responsible for the lateral gene The first mobile elements described were the transfer. The origin of these genes is not known, simple insertion sequences (IS). General features but recent analyses of available data suggested of insertion sequences are that they encode no that the gene cassettes might be the ancient struc- function except of those genes involved in their tures (RECCHIA &HALL, 1997). Several transpos- own mobility (CAMPBELL et al., 1979). These in- able elements, carrying antibiotic resistance genes, r clude the factors required in cis recombination, in e.g. Tn1 [Ap ](HEDGES &JACOBS, 1974); Tn3 r r particular the recombinationally active DNA se- [Ap ](KOPECKO &COHEN, 1975); Tn5 [Km ] r r quences that define the ends of the element accom- (BERG et al., 1975); Tn7 [Tmp ,Str](AMYES & r panied with enzyme – transposase, which recog- SMITH, 1978); Tn9 [Cm ](GOTTESMAN &ROS- r nises and processes these ends. The majority of ISs NER, 1975); Tn10 [Tc ](FOSTER et al., 1975) were exhibit the short terminal inverted repeates (IRs) recognized almost simultaneously in the mid 1970s sized between 10 and 40 bp. The sequence, encod- as the natural components of R-factor plasmids ing the transposase gene is often located partially executing their ability of transposition. The re- within these IRs accompanied by the upstream combinant DNA methods became widely avail- promoter with the conventional IR sequences. This able shortly after the discovery of resistance trans- arrangement provides the binding mechanism for posons and the resistance genes were frequently autoregulation of synthesis. The sequence-specific incorporated into the plasmid cloning vectors, cur- DNA binding activities of transposase are gener- rently common in use. ally located in the N-terminal region, while the Conjugative transposons are the important catalytic domain is often localized towards the determinants of antibiotic resistance, mainly in C-terminal region. An additional characteristic of Gram-positive bacteria. They are remarkably pro- many transposases is the capacity to generate mul- miscuous providing the conjugation between bac- timeric forms, essential for their activity. Another teria, especially those of different species and gen- general feature of IS elements is the generation era. Transposon-promoted conjugation reminds of short directly repeated sequences (DRs) of the the F plasmid one, thus only the single strand of target DNA flanking the IS during the insertion. the transposon DNA is transferred from donor to The length of DRs, usually between 2 and 14 bp, recipient. The mechanism of recombination dur- is characteristic for each element (MAHILLON & ing the conjugative transposition differs from that CHANDLER, 1998). of other transposons, as it was shown for exam- Early studies have shown two ways of inter- ple by the absence of duplications of the target molecular insertion mechanisms – the conserva- sequence upon the integration (BRINGELL et al., tive and replicative transposition. The conserva- 1992). The site-specific recombinases, encoded by tive (nonreplicative) transposition known in Tn10 the conjugative transposons, belong to the inte- and Tn5 (originated from lambda phage) was de- grase family. Alike the phage lambda integrase, the scribed as a transposition of element, separated integrase of Tn916 has two DNA-binding domains from vector DNA, by a double-strand break at that recognize different sequences, one within the each end. This insertion into the target DNA se- ends of the element and one that includes target quence is realized without prior replication, and DNA-not specific sequences, but apparently con- 310 sist of bent DNA. The similarity between the con- the gene and large-scale genome mapping, for the jugative transposons and phage lambda is striking in vivo cloning and the cloned DNA sequencing and suggests that both use the same mechanism (BERG &BERG, 1996). of recombination, with the exception of the re- DNA transposition studies up to date have combining sites that are nearly always different in involved strictly in vivo approaches, in which the the conjugative transposition (SCOTT &CHURCH- transposon of choice and the gene encoding trans- WARD, 1995). The first step in the conjugative posase, the enzyme responsible for transposition, transposition of Tn916 is the excision from donor had to be introduced into the cell together. How- DNA molecule, followed by the circularisation of ever, the in vivo systems have many technical transposon and its transfer to a new host. The limitations. Therefore, a large number of in vitro studies have demonstrated that in Gram-positive transposition systems for Tn5,Tn7,Mu,Himar1 hosts, both the Xis protein and the site-specific and Ty1, which bypass many limitations of in vivo recombinase Int are required for the excision of systems, have been constructed. For this purpose Tn916 (RUDY et al., 1997). Neither protein alone also, a technique for transposition that involves is the rate limiting for excision, but overexpression the formation in vitro of release Tn5 transposition of Int and Xis together
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