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Home , Insertion sequence, Plasmid, Tn10, Transduction (genetics), Transposon mutagenesis

Biologia, Bratislava, 59/3: 309—318, 2004 REVIEW

Transposons – the useful genetic tools

Miriam Vizváryová1 &DankaValková2*

1St. Elizabeth Institute, Heydukova 10,SK-81250 Bratislava, Slovakia 2Department of Molecular , 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 ). Mobile DNA elements, originally discovered in more than fifty years ago, have become the indispensable tools for bacterial : 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 , fu- sion and mapping techniques as well as DNA sequencing strategies. Recent applications have extended transposition-based techniques to the analysis of , , -DNA complexes and proteomes. Key words: transposon, transposition, in vivo , functional genomics.

Abbreviations: ATC, altered target specificity; DR, direct repeat; IR, ; IS, sequence; oriT, origin of transfer; Tn, transposon; Tnp, protein; Apr,Kmr,Tmpr,Strr,Cmr,Tcr,Err,resistancetofol- lowing : Ampicillin, Kanamycin, Trimetoprim, Streptomycin, Chlo- ramphenicol, , 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 . 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 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 (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. (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 and Many of the acquired antibiotic resistance heavy metal resistance. , 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 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 , the mobile and . 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 – 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 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- 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 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 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 ) 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 resulted in the increased complexes, followed by the introduction of com- excision. After excision, the frequency of Tn916 plexes into the target cell of choice by electropo- circle formation was found to be the same as the ration (GORYSHIN et al., 2000) was developed. frequency of repair of donor DNA molecule. This The strategy useful for transposon delivery suggested the close similarity of a single reaction depends on the target strain, on whether the tar- in both molecules (MARRA &SCOTT, 1999). get is the bacterial , a phage, or plas- Lots of transposons are the normal con- mid. (BERG &BERG, 1996). For chromosomal tar- stituents of the most bacterial genomes and of gets, transposon is usually delivered on phage, sui- many extrachromosomal plasmids and bacterio- cide vector or F factor. Usually, the suicide vectors phages. The worldwide research indicates that are used, when a variety of suicide strategies based these DNA insertion elements play a special evolu- on mutant plasmids are unable to replicate under tionary function (CAMPBELL, 1981; KIDWELL & the special condition, for example – high temper- LISCH, 2000). ature. Delivery vehicle determines the choice of tar- Transposons as the molecular genetic tools get molecule. are the most conve- nient type of delivery vehicles for insertion into Recently, there took place the explosive devel- the bacterial chromosome. The phage carrying a opment of in vitro DNA technology. Joining of transposon can be introduced into the host cell DNA segments in vivo, however, has been possi- under the conditions, disabling the phage genome ble for a number of years. Transposons have been replication, cell lysis, or even its stable integration evolved as the natural tools for genetic engineer- into the host cell. Lambda vehicles are used for ing (BUKHARI, 1977). The most widely used con- the isolation of Tn10,Tn5 and Mu insertions. Mu structs were derived from the insertion sequences, is also used directly. IS containing composite elements transposons, or Nonconjugative multicopy plasmids and bac- from bacteriophages. The classical applications of teriophages are also the delivery vehicles of choice transposable elements in bacterial genetics can be for the isolation of insertions. The specific plas- distinguished, based on the type of insertion. In mid vehicles have also been constructed for this general, the transposition from one DNA molecule purpose. to another is usually utilized for the stable mainte- In the case the target molecule is phage or nance of genetic marker present on the transposon conjugative plasmid, the transposon delivery vehi- as the source of selectable marker; on the other cle can be utilized, any type of molecule or repli- hand the transposons are often used as the inser- con other then the target itself. General features tion mutagens generating deletions and inversions of transposon derivatives are important if the pri- during their transposition. mary goal is the isolation of stable insertion into Additional application of transposons is a target gene or region of interest. based on their ability of the portable reporter For stability it is always preferable to use genes expression control, the transcriptional-trans- a mini-transposon construct whenever other con- lational fusions as well as for the transpositional siderations permit. Mini-transposons are referred fusion. Transposable elements were acquitted as as transposons that do not contain a transposase the useful tools for also for gene within their boundaries and are generally

311 smaller than a corresponding wild-type transpo- tion and selected with the appropriate antibiotic. son. All these systems possess a number of useful fea- tures: the variety of antibiotic markers (Err,Cmr, Transposon Tn10 and its derivatives Kmr or Tcr); the polylinker containing the restric- tion sites for rare-cutting endonucleases to facili- Tn10, the composite bacterial transposon com- tate the physical mapping of chromosomal inser- prising of two IS10 elements (R and L) plus in- tions; the mutant transposase that confers relax- ternal sequences including tetracycline resistance, ation in insertion specificity and positioning of the can move into and out of or plasmids transposase-encoded gene outside of the transpos- in a non-replicative fashion (HANIFORD &CHA- ing segment to ensure the stability of insertions CONAS, 1992; KLECKNER et al., 1996). Recently, once isolated. the complete nucleotide sequence of Tn10 has been The new derivative, based on a mini-trans- determined (CHALMERS et al., 2000). Using Tn10 poson Tn10 named NKBOR, has been constructed for generation of by transposon inser- (ROSSIGNOL et al., 2001). This mini-transposon tion can be a powerful analytical technique. His- contains a conditional R6K-suicide vector that torically derivatives of bacterial transposon Tn10 permits the random insertions into the chromo- were described that were useful for defining the some of Gram-negative bacteria and the subse- functional limits and regulatory sites of bacterial quent rapid cloning of sequences flanking the in- genes (WAY et al., 1984). sertion site in E. coli. Wild-type Tn10 preferentially inserts into the HARE et al. (2001) have developed the in vivo so-called hotspots. Potential sites of Tn10 inser- genetic foot-printing system based on Tn10 trans- tion cannot be predicted, but generally four from poson for E. coli, a model bacterium for rapid dis- six base pairs in the consensus sequence are GC covering the genes that affect the cell fitness under pairs. Mutant IS10 transposase strains with al- the variety of growth conditions. This system en- tered target specificity (ATS) exhibit significantly ables the high frequency of randomly distributed lower degree of insertion specificity than a wild transposon insertions, utilizing the conditionally type (HALLING &KLECKNER, 1982). regulated Tn10 transposase, with relaxed sequence A lot of Tn10 derivatives, useful for various specificity and the conditionally regulated repli- types of mutagenesis carried on plasmid and/or con for the vector, containing the transposase and phage vehicles, have been constructed by KLECK- mini-Tn10 transposon with an outwardly oriented NER et al. (1991). These constructs named NK promoter using the lambda delivery can be divided into several types (Fig. 1): (i) system. those carrying the wild-type Tn10 (101), bear- ing an IS10 Left and Right, the internal sequence Transposon Tn5 and its derivatives and tetracycline resistance genes; (ii) derivative 102, which contains the ats and trans- Wild type of Tn5 is a 5700 composite posase gene fused to the strong, IPTG inducible element, in which a pair of simpler mobile ele- Ptac promoter useful tool for complementation in ments, e.g. 1534 bp insertion sequences IS50Land trans mini-Tn10; (iii) derivatives 103–108 mini- IS50R, are present in an inverted orientation with Tn10 with ATS are small (400–3000 bp) and can a bracket of central region that contains genes be used for stable insertions, because they do not encoding resistance to kanamycin (kan) and/or carry a transposase gene (these are available with streptomycin. Tn5 transposes with high frequency a variety of selectable markers); and (iv) finally the and inserts into many sites, including a small num- mini-Tn10 construct, which generates the transla- ber of hotspots. Alike several other bacterial ele- tional fusion of lacZ to target gene and the trans- ments, Tn5 generates a direct 9-bp duplication of lation fusion to kan gene. the target sequence at its site of insertion. One of Later the plasmid-based vehicles were de- the two IS elements in Tn5,IS50Rencodesacis- scribed, which can be used for delivery of IS10- acting protein, transposase, that is necessary for derived transposons into Gram-negative and also IS50 and Tn5 movement. Transposase is thought capable to replicate in Gram-positive bacteria to act directly by binding to distinctive 19-bp nu- (MAHILLON &KLECKNER, 1992). All the deriva- cleotide sequences near the ends of its recognized tives described above use the standard delivery ve- elements (JOHNSON &REZNIKOFF, 1983). IS50L hicles based on bacteriophage lambda or plasmid differs from IS50R in that it contains the promoter pBR322. The transposons carried on plasmid can used for expression of kan gene in Tn5’s central re- be introduced by or transforma- gion and also it possess an ochre allele of the trans-

312 Fig. 1. Genetic and physical maps of the different mini-Tn10 derivatives and their using. Cleavage recognition sites for restriction are: Bc, BclI; Bg, BglII; C, ClaI; H, HindIII; R, EcoRI; Xb, XbaI. Structure of each transposon is drawn to scale. posase (tnp) gene. Both the kan promoter and the able to dissociate itself from a DNA product; mutant tnp allele arose from a substitution of sin- Tn5 transposes by a conservative “cut and paste” gle nucleotide pair 112 bp form IS50’s inside end mechanism; Tn5 release from the donor back- (BERG &BERG, 1983; BERG et al., 1984). bone involves the precise cleavage of both 3’ and Tn5 has become the model for development 5’ strands at the ends of the specific end se- of an efficient in vitro transposition system. The quences (ZHOU &REZNIKOFF, 1997; GORYSHIN key component of such system was the use of hy- &REZNIKOFF, 1998). peractive mutant transposase. The inactivation of The Tn5 transposition process involves the the wild type transposase is likely to be related to following steps: (i) binding of transposase mono- the low frequency of in vivo transposition. mers to the 19 bp end sequences; (ii) oligomer- The in vitro experiments have demonstrated ization of the end-bound transposase monomers, the following: the only required macromolecule for forming a transposition synaptic complex; (iii) the most of steps in Tn5 transposition is trans- blunt end cleavage of the transposition synaptic posase; the specific 19-bp Tn5 end sequences, complex from adjoined DNA, resulting in forma- and the target DNA; transposase may not be tion of the released transposition complex or trans-

313 poson; (iv) binding to target DNA; and (v) strand plasmid vehicles and as chromosomal insertions transfer of transposon 3’-ends into a staggered 9 to extend the range of targets for Tn mutagen- bp target sequences (ZHOU &REZNIKOFF, 1997; esis. Single EcoRI sites at the ends of these trans- GORYSHIN et al., 2000, REZNIKOFF, 2003). posons have been proved as the most useful for The Tn5 in vitro transposition system pro- physical mapping, for the generation of new EcoRI vides a highly efficient one step reaction, contain- sites in cloning experiments, for end-labelling and ing two macromolecular components: a hyperac- for sequencing of DNA adjacent to the insertion tive form of Tn5 transposase (Tnp) and DNA (UBBEN &SCHMITT, 1986). containing two inverted 19 bp Tnp recognition Derivatives of Tn1721 are useful because of sites. Synthetic transposons have to contain an their properties, i.e. random insertion and gener- ori, an antibiotic resistance gene marker, a mul- ation of transcriptional fusions at the site of in- ticloning site and two hyperactive end sequences. sertion: transposable promoters (Tn1735) carry a This transposon-plasmid, containing no target se- strong, inducible Ptac promoter that turns on ad- quences should be incubated in the presence of jacent (cryptic) genes; and transposable promoter purified transposase protein and transformed into probes (Tn1736,Tn1737) carrying promoterless desired target strain to undergo transposition genes encoding chloramphenicol acetyl (YORK et al., 1998). Construction of such mini- or β-galactosidase frequently used for accurate de- Tn5 vector – pUT was developed by LORENZO termination of external promoters. These elements et al. (1998). Tn5 is a composite transposon; its are available with four different selectable resis- mobility is determined by two insertion sequences tance markers and on conjugative, temperature- (IS50L and IS50R) flanking the DNA region en- sensitive and multicopy plasmid vehicles. Experi- coding the Kmr genes. Interestingly, as in Tn10, ments were described that demonstrate the advan- the transposase determined by IS50R(tnp gene) tage of random insertions for various genes expres- is still functional, even if the gene is artificially sion and for the gene regulation studies (UBBEN placed in cis to the cognate terminal sequences. &SCHMITT, 1987). Mini-Tn5 possesses all elements essential for trans- position (IS terminal sequences and tnp gene) and Mu and mini-Mu derivatives use for genetic delivering system into the target strain, based on analysis plasmid, e.g. R6K, which requires a specific repli- cation protein PI, maintained only in host strains. has been described as a temper- Some mini transposon vectors have the origin of ate phage, which upon lysogenization generated replication as well as the origin of transfer (oriT) mutation in host with the highest transposition from F factor (or pRK2 in pUT plasmid). The frequency and the most random insertion speci- tnp gene is lost shortly after insertion and mini- ficity of any known transposons. A striking differ- transposons are anchored preventing the DNA re- ence between Mu and other temperate phages is arrangements or any form of genetic instability. that the Mu genome integrates in the host chro- mosome whether it enters the or the Tn1721 family transposons and their lysogenic state. Its transposition requires the pres- derivates ence of cis-andtrans-acting Mu DNA sequence, especially the left end of Mu phage. Mu was Tn1721 transposon as well as Tn501 and Tn21 be- the first with the evidence long to a subgroup of Tn3 family, although their of replicative transposition mechanisms. Products sequence homology is relatively low. Transposition of three genes, e.g. c, ner and MuA,whichen- of Tn3-like elements is characteristic for involving codes transposase, control transposition. Product the formation of co-integrates, catalysed by trans- of MuB gene is required for transposition in high posase and co-integrates resolution catalysis by re- level. Packing proceeds from the pac sites – the left solvase. The second step is strictly site-specific and end towards the right end, by the head full system requires two directly oriented copies of the resolu- up to 35–58 kb (KAHMANN &KAMP, 1979). tion sequence in the co-integrate (SCHMITT et al., Bacteriophage Mu based vectors, used for ge- 1981; ROGOWSKY &SCHMITT, 1985). netic engineering purpose, harbour a cis action Originally isolated Tn1721 transposon con- terminal DNA sequence, necessary for transposi- tains tetracycline resistance; many of new deriva- tion, the pac site and a selectable marker. Most tives carrying resistances to chloramphenicol, te- of derivatives contain a thermo-sensitive (ts) al- tracycline, kanamycin and streptomycin were de- lele of repressor (FAELEN &TOUSSAINT, 1976). scribed. These elements are provided on various Mu derivatives, which can still grow as phage,

314 are designated by the letter “p”, indicating that economical (skipping DNA isolation, restriction they can form plaques (e.g., MupAp1); the let- and ligation steps), the main advantage being the ter “d” indicates that they are defective and un- ability to clone relatively large regions of a chro- able to grow as phages, but they can cause the mosome and those bearing toxic or harmful genes rearrangements, typical for these transposable ele- that are difficult to clone using the in vitro sys- ments (e.g., MudI1). Mu derivatives that can form tem (STUCHLÍK et al., 1993a; VIZVÁRYOVÁ et al., transcriptional or transcriptional-translational fu- 2002). sions are usually designated MudI and MudII, re- spectively (GROISMAN, 1991). Mu elements can be Transposons used in post genomic era introduced into bacteria by , conju- gation, or transformation. Transduction has been In this post genomic era, complete DNA sequences widely used because Mu has a very broad host of many microbial genomes are available, but range and because all derivatives possess the pac nearly half of the putative genes lack identifiable sites. Mu lysates are often prepared from dou- functions. Identification of genes with their func- ble lysogens, harbouring both the defective Mu tion is necessary to convert the sequence data into derivatives to be selected for and a helper Mu meaningful biological information. phage that will complement the Mu derivatives for revealed a powerful the morphogenic functions. Transformation has technique for the minimal Mycoplasma genitalium been usually used to introduce mini-Mu plasmids genome estimation (HUTCHINSON et al., 1999) as (GROISMAN et al., 1984). well as for the identification of bacterial virulence Transposon mutagenesis has become the pro- factors (AKERLEY et al., 1998; GUO et al., 2001; cedure of choice to isolate mutants – inactivation GAO et al., 2003). of gene, mutagenesis of open reading frames, tran- A new family of transposons with a broad scriptional and translational fusions (in vivo mu- host range and short recognized sequence has been tagenesis) or in vivo cloning and mapping DNA. developed, based on the eukaryotic transposons, For chromosome as a target it usually can be in- providing the identification of essential genes by teresting to obtain the stable insertion by per- in vitro transposition – marine mutagenesis. forming the mutagenesis with a derivative that is So-called mariners are the widespread an- defective for transposition but that can be com- imal transposons, present in many different in- plemented in trans. This can be accomplished by sect species. Mos1 (from fruit fly Drosophila using phage that only transpose in suppressor melanogaster,ROBERTSON &LAMPE, 1995) and + backgrounds, by using the phage that is MuA Himar1 (from horn fly Haematobia irritans, LAM- and co-infecting with a phage that carries the PE et al., 1996) are among the best-studied MuB+ gene in trans or by using the MuA−B− mariner transposons targeting the AT dinucleo- derivatives. Mu phage is especially suitable for tide recognition site that are able to insert into the in vivo cloning, because it transposes hun- diverse genomes, including both and dreds of times as it replicates when derepressed (FADOOL et al., 1998; GUEIROS- for its lytic functions (BREMER et al., 1984). Many FILHO &BEVERLEY, 1997). mini-Mu derivatives with lac fusion elements were MaT, another clade of mariner transposons, prepared for gene structure and expression stud- originated from housefly Musca domestica, is ies (CASTILHO et al., 1984). These can be useful flanking the gene involved in organophosphate in- for thein vivo cloning so far, because they pos- secticide resistance, but highly similar homologue sess plasmid high-copy (or low-copy), broad-host- was found also in silkworm moth Bobyx mori,and range replicons, different resistance markers, some even 16 copies of maT sequence were found in the of them also an oriT(GROISMAN &CASADA- genome of Caenorhabditis elegans (CLAUDIANOS BAN, 1986; STUCHLÍK et al., 1993b; OSUSKÝ et et al., 2002). Since the identical mariners were iso- al., 1994). Low-copy mini-Mu vectors were used lated and sequenced in several different species, successfully for cloning of toxic genes (LACZAOVÁ they are supposed to take part in primary selec- et al., 1995; GRONES et al., 1996; BURIAN et al., tive sequence evolution spread by the horizontal 1998; TU et al., 2001). See also Fig. 2 for more gene transfer (LAMPE et al., 2003). details. Despite the fact that the palette of in vitro The derivatives of Mu bacteriophage thus be- techniques has been growing rapidly together with came useful molecular biology tools both for the the genomics generated data, the use of in vivo in vivo and in vitro techniques. Mini-Mu phage techniques, including transposons will still remain cloning methods have many advantages, besides the powerful tool of functional genomics studies.

315 Fig. 2. Genetic and physical maps of the different mini-Mu derivatives and their using. Cleavage recognition sites for restriction enzymes are: B, BamHI; E, EcoRI; P, PstI; S, SalI; V, PvuII, X, XhoI. The other sites are as in Fig. 1. Structure of each mini-Mu is drawn to scale.

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Received June 4, 2004 Accepted November 20, 2003

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