| Nucleic Acids Research | Pages |
An efficient and accurate integration of mini-Mu transposons in vitro: a general methodology for functional genetic analysis and molecular biology applications
Introduction
Materials And Methods
Proteins and reagents
DNA and oligonucleotides
DNA techniques, mini-transposons and Escherichia coli strains
In vitro transposition reactions and biological selection of integrants
Target site selection
Generation of the selected plasmid pool
Localization of functional DNA region by restriction analysis
Genetic footprinting
Results
Discussion
Acknowledgements
References
An efficient and accurate integration of mini-Mu transposons in vitro: a general methodology for functional genetic analysis and molecular biology applications
Received February 8, 1999; Revised and Accepted May 6, 1999
ABSTRACT Transposons are mobile genetic elements and have been utilized as essential tools in genetics over the years. Though highly useful, many of the current transposon-based applications suffer from various limitations, the most notable of which are: (i) transposition is performed in vivo, typically species specifically, and as a multistep process; (ii) accuracy and/or efficiency of the in vivo or in vitro transposition reaction is not optimal; (iii) a limited set of target sites is used. We describe here a genetic analysis methodology that is based on bacteriophage Mu DNA transposition and circumvents such limitations. The Mu transposon tool is composed of only a few components and utilizes a highly efficient and accurate in vitro DNA transposition reaction with a low stringency of target preference. The utility of the Mu system in functional genetic analysis is demonstrated using restriction analysis and genetic footprinting strategies. The Mu methodology is readily applicable in a variety of current and emerging transposon-based techniques and is expected to generate novel approaches to functional analysis of genes, genomes and proteins.
INTRODUCTION
Transposons, mobile DNA elements originally discovered by Barbara McClintock in maize (1), have become indispensable tools in the genetics of both prokaryotic and eukaryotic organisms (2,3). Classical in vivo transposition applications include insertional mutagenesis, gene fusion and mapping techniques as well as DNA sequencing strategies (4-8). Recent applications have extended transposition-based techniques to the analysis of genomes, proteins and protein-DNA complexes (9-16).
The mechanism of DNA transposition follows a common scheme with some variation in details depending on the element (17,18). In the simplest of cases, a mobile element encodes a transposase protein that recognizes the transposon ends, forms a higher order protein-DNA complex, called a transpososome, and then facilitates transposition by catalyzing DNA cleavage and joining reactions (18-21). Bacteriophage Mu, which uses DNA transposition to replicate its genome, is one of the best-characterized mobile genetic elements and the first for which an in vitro transposition reaction was established (22). In vivo and in certain reaction conditions with plasmid substrates in vitro the Mu DNA transposition reaction is relatively complex (Fig. 1A). It involves: (i) the catalytic MuA transposase; (ii) the transposon end sequences including MuA binding sites essential for transpososome architecture; (iii) a certain topology of the transposon-containing DNA substrate; (iv) a number of accessory protein and DNA cofactors. Of the cofactors the most critical are the host-encoded DNA bending protein HU, the phage-encoded protein MuB, which affects target choice, and the transpositional enhancer located internally in the phage genome (for reviews see 17,23). With modified DNA substrates and reaction conditions in vitro, however, the requirements for transposition are relaxed substantially. In a minimal set-up, the MuA transposase and appropriate DNA substrates are the only macromolecular components required (24,25).
A
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B
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Figure 1. (A) Relationship between the Mu DNA transposition reaction with plasmid substratesin vitro (above) and the simplified reaction used in this study (below). In both cases a tetramer of MuA transposase first forms, with the transposon ends, an active DNA transposition complex, a transpososome. The transpososome then executes the donor cleavage and strand transfer steps of transposition to yield the transposition intermediate (note that the donor cleavage step is bypassed by the use of precut donor DNA in the current method). Then, after disassembly of the transpososome, the intermediate is processed by host factors. Depending on the reaction pathway (replication or repair) the end products are co-integrates or simple insertions, respectively. Critical protein components required for separate steps of transposition are indicated. The DNA 3[prime]-ends are marked with dots. (B) Mini-transposon constructions between EcoRI and HindIII sites in plasmid pUC19 with relevant joint sequences shown. Long arrows, selectable marker genes (supF and cat); short arrows, primer binding sites. Rectangles denote 50 bp of DNA derived from the Mu R-end (24). Below is shown the sequence of the left end of mini-Mu transposons as well as the flanking region in pUC19. In the right ends the same sequence is present in inverted orientation and the EcoRI site is replaced by a HindIII site. R1 and R2 (boxed sequences) denote MuA binding sites (24).
In this paper we describe a methodology for functional genetic analysis and molecular biology applications based on the bacteriophage Mu DNA transposition reaction modified for such purposes (Fig. 1A). The transposon tool described utilizes a highly efficient and accurate in vitro reaction with relatively even distribution of target sites. The methodology is versatile and expected to be highly useful in various molecular genetic tasks. These include not only traditional transposition applications but also novel approaches for the analysis of genes, genomes and proteins.
MATERIALS AND METHODS
Proteins and reagents
MuA proteins were purified in collaboration with Finnzymes (Espoo, Finland) essentially as described (26,27). Restriction endonucleases, [beta]-agarase, T4 polynucleotide kinase (PNK), and Vent DNA polymerase were from New England Biolabs. Dynazyme II DNA polymerase and calf intestinal alkaline phosphatase (CIP) were from Finnzymes. Taq DNA polymerase and DNase I were from Promega, T4 DNA ligase from Life Technologies and bovine serum albumin (BSA) from Miles. All enzymes were used under the reaction conditions recommended by the supplier. [[gamma]-33P]ATP (1000-3000 Ci/mmol) and [[alpha]-33P]dATP (1000-3000 Ci/mmol) were from Amersham. Agaroses were from FMC Bioproducts.
DNA and oligonucleotides
Plasmids pCDNA1 and pBC SK+ were from Stratagene and pUC19 from Life Technologies. Supercoiled pUC19 was prepared using the Qiagen plasmid isolation kit and purified by CsCl gradient centrifugation. The nicked circular form was then prepared by a limited digestion with DNase I. The linear form was made by SapI digestion. All three forms of pUC19 were purified by electrophoresis through a SeaPlaque agarose gel and recovered by electroelution using a Biotrap apparatus (Schleicher & Schuell). The oligonucleotide primers were: HSP4, 5[prime]-TCACAGCTTGTCTGTAAGCGGATGC; HSP5, 5[prime]-TGAA-ATACCGCACAGATGCGTAAGG; HSP6, 5[prime]-CATTTAT-CGTGAAACGCTTTCGCG; HSP19, 5[prime]-CCGCTGTAAAGTGTTACGTTG; HSP20, 5[prime]-CGAAAGACCGCGGTCCAGCTG; HSP55, 5[prime]-GCAAGGCGATTAAGTTGGGTAACGCC; HSP90, 5[prime]-AAGCCTGGGGTGCCTAATGAGTG; HSP91, 5[prime]-CTGG-GCTGTGTGCACGAACC; HSP92, 5[prime]-GCCTGACTCCCCG-TCGTGTAGA; HSP93, 5[prime]-GGTCCTCCGATCGTTGTCAGAAG; HSP94, 5[prime]-GGGCGACACGGAAATGTTGAATA; HSP164, 5[prime]-CGCCAGGGTTTTCCCAGTCACGAC.
DNA techniques, mini-transposons and Escherichia coli strains
Standard DNA techniques were performed as described (28). DNA sequencing was done using an ABI 377 DNA sequencer (Perkin-Elmer). The polylinker between EcoRI and HindIII sites in pUC19 was replaced with mini-transposon constructs by standard PCR (using Vent DNA polymerase) and cloning procedures (Fig. 1B). The selectable markers between BamHI sites in supF-Mu (supFamber suppressor tRNA) and cat-Mu (chloramphenicol acetyltransferase gene, cat) were derived from plasmids pcDNAI and pBC SK+, respectively. Mini-transposons were isolated from vector plasmids by BglII digestion (leaving four nucleotide 5[prime]-overhangs) and purified using the anion exchange columns Gen-Pak FAX from Waters (for supF-Mu) and TSK-gel DNA-NPR from Tosohaas (for cat-Mu). When required, the mini-transposon 5[prime]-ends were dephosphorylated with CIP, after which the DNA was labeled with [[gamma]-33P]ATP and PNK. SupF-Mu and cat-Mu phenotypes were detected in strains MC1061/P3 (Invitrogen) and DH5[alpha] (Life Technologies), respectively. The plasmid P3 in the former strain contains an amber mutation in both the ampicillin and tetracycline resistance genes. Cloning host was strain DH5[alpha].
In vitro transposition reactions and biological selection of integrants
Standard reactions (25 µl) contained 10 nM donor DNA, 250 ng target DNA, 224 nM (0.4 µg) MuA, 25 mM Tris-HCl, pH 8.0, 100 µg/ml BSA, 15% (w/v) glycerol, 0.05% (w/v) Triton X-100, 126 mM NaCl and 10 mM MgCl2. Reactions were carried out for 1 h at 30°C unless otherwise indicated. Reactions with a short Mu R-end DNA fragment as the donor were carried out as described (25). Transposition reaction products were analyzed by electrophoresis as described(25) on a 1% SeaKem HGT agarose gel in 1× TAE buffer. The EtBr-stained gel was photographed on Polaroid 665 film. Quantification of reaction components from negatives was made with an AlphaImager digital analysis system (Alpha Innotech Corporation). For autoradiography, the gel was dried onto Whatman DE-81 paper and the reaction products were visualized using a Fuji BAS 2000 phosphorimager. Autoradiograms were quantified with the Tina 2.0 program (Raytest). For biological selection of integrants, the transposition reaction products were phenol and chloroform extracted, ethanol precipitated and resuspended in 25 µl of TEN (10 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 50 mM NaCl), of which 1 µl was transformed into competent E.coli cells(28).
Target site selection
Three microliters from an in vitrotransposition reaction (supF-Mu donor, pBC SK+ target, Fig. 3B) was used as a template in a PCR reaction (volume 50 µl). The primer pairs were HSP6/HSP164 and HSP6/HSP55, of which HSP164 and HSP55 were 33P-labeled. Five units of Taq DNA polymerase were used and 18 cycles of amplification were performed with 1 min at 96°C, 1 min at 65°C and 1 min at 76°C. Target site selection with different forms of pUC19 was also studied by PCR. One microliter from an in vitro transposition reaction was used as a template in amplification (volume 50 µl). The primer pair used was HSP6/HSP5, of which the pUC19-specific HSP5 was 33P-labeled. Two units of Dynazyme II DNA polymerase were used and 30 cycles of amplification were performed with 45 s at 95°C, 1 min at 65°C and 1 min at 72°C (with an additional 4 min at 72°C in the last cycle). PCR products were analyzed by denaturing 7 M urea-6% polyacrylamide gel electrophoresis (urea-PAGE) and autoradiography as described (25).
Generation of the selected plasmid pool
Five transposition reactions (25 µl each) were performed with cat-Mu donor (1 nM), MuA (0.2 µg) and pUC19 target (250 ng) for 4 h as described above. The reactions were pooled and sequentially phenol and chloroform extracted, ethanol precipitated and resuspended in 25 µl water. Several 1 µl aliquots were electroporated into 25 µl of electrocompetent DH5[alpha] cells prepared as described (29) using 1 mm cuvettes in a Bio-Rad Genepulser II with the following settings: capacitance 25 µF, voltage 1.8 kV and resistance 200 [Omega]. After electroporation, 1 ml Luria medium per aliquot was added and bacteria were grown for 1 h at 37°C, collected by centrifugation and plated on LB-chloramphenicol (5 µg/ml) selection plates. Approximately 3 × 104 colonies were obtained, pooled and then grown in LB-chloramphenicol (5 µg/ml) medium for 2 h, after which the plasmid DNA from the pool was isolated.
Localization of functional DNA region by restriction analysis
The selected plasmid pool was digested with BfaI and AseI and analyzed in a 0.65% SeaPlaque agarose gel in 1× TBE buffer followed by EtBr staining or Southern blotting. The primary transposition reaction product was used as the unselected pool. It was isolated from a 0.8% SeaPlaque agarose gel using [beta]-agarase and then digested with BfaI. Southern hybridization was carried out on Hybond membrane (Amersham) with [alpha]-33P-labeled (Random Primed; Boehringer Mannheim) cat-Mu as a probe. Quantification of hybridization signal on the membrane was done using a phosphorimager (see above).
Genetic footprinting
DNA from the selected plasmid pool (50 ng) and an equivalent amount of DNA directly from a transposition reaction (unselected pool) served as templates in PCR (volume 100 µl). Six 33P-labeled pUC19-specific primers were used in combination with Mu end-specific primer (Fig. 5). Dynazyme II DNA polymerase (2 U) was used and 17 cycles of amplification were performed with 45 s at 96°C, 1 min at 65°C and 1 min at 72°C. PCR products were analyzed by urea-PAGE as above.
RESULTS
We constructed two selectable marker-containing mini-Mu transposon substrates, supF-Mu and cat-Mu (Fig. 1B). Labeled supF-Mu was used as a donor in the in vitro transposition reaction with plasmid pBC SK+ as a target (Fig. 2A). A dominant reaction product was formed efficiently (lanes 4 and 5) and it represented integration of a single donor molecule into the target plasmid (identity shown earlier; 24). Target molecules with two insertions could also be detected. As expected, when the concentration of donor DNA was reduced, the yield of reaction products decreased and the ratio of the single integration product to the double integration product increased (lane 5 and data not shown). In order to distinguish between one-ended and two-ended integrations the reaction products were cleaved with restriction enzyme AvaII (Fig. 2B). As expected (30), since MuA is active as a tetramer, mixtures of MuA and active site mutant MuAE392Q yielded reaction products that were derived from both one-ended and two-ended integrations (lanes 4, 6 and 8). However, when only wild-type MuA was used, essentially all reaction products were derived from two-ended integrations (lane 2). Thus, under the conditions used, the main reaction product was derived from two-ended integration, involving a single supF-Mu donor molecule, into the target.
A
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B
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Figure 2. (A) The in vitro transposition reaction utilizing 33P-labeled supF-Mu donor DNA and unlabeled plasmid pBC SK+ as the target DNA. Potential reaction products and their origin are shown schematically. Even though not depicted, multiple integrations into one target are also possible, as are the reaction products (intermolecular or intramolecular) utilizing the donor DNA as a target. (Left) An EtBr-stained agarose gel. (Right) Autoradiogram of the gel. Lane 1, linearized pBC SK+; lane 2, reaction without MuA; lane 3, reaction without donor DNA; lane 4, reaction (10 nM donor); lane 5, reaction (1 nM donor). D, donor; TO, nicked circular target; TC, supercoiled target; TL, linear target; IS, single integration product; ID, double integration product. Reactions did not yield a detectable level of a reaction product involving two Mu end sequences on two separate donor molecules; the formation of such a product was dependent on the donor DNA concentration and could be detected at higher donor DNA concentrations (23; data not shown). (B) Distinction between one-ended and two-ended (i.e. concerted) integrations by AvaII digestion of the reaction products. To induce one-ended integrations in control reactions we mixed wild-type MuA and the active site mutant MuAE392Q, defective in the chemical steps of transposition (30). The scheme of the analysis and product analysis by agarose gel electrophoresis and autoradiography. Integration products as in (A); ACL, ACS and AL, AvaII digestion products.
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B
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Figure 3. (A) SupF-Mu integration sites in plasmid pBC SK+ (see http://www.stratagene.com for the sequence, notation and coordinates). Selection was on LB plates containing ampicillin (25 g/ml) and tetracycline (8 g/ml). In each integration site the coordinate of the first 5[prime]-nucleotide of the duplicated target sequence is shown. The orientation of the supF-Mu transposon is indicated by + (direction of supF transcription the same as that of the cat gene) or with - (direction opposite). +/- denotes two independent integrations into the same site but with opposite orientation. (B) A PCR-based analysis of target site selection. Each band in the autoradiogram corresponds to integration at a specific phosphodiester bond. The intensity of the bands corresponds to the relative frequencies of integration into particular sites. As PCR controls, we used reaction mixtures from which either MuA or donor DNA was omitted. To further verify the specificity of the amplification, we used two different target plasmid-specific primers, the hybridization sites of which are located 22 nt apart from each other in the target DNA (note the corresponding shift in the patterns between lanes 3 and 6). The triangles and arrows denote the transposon insertions and the primers used, respectively. The radioactive labels are shown by asterisks. Reaction products were analyzed on a 6% urea-polyacrylamide gel. Primer pairs used are shown on the top and approximate lengths of the PCR products on the right.
The integration events into target plasmids were scored by introducing products from the in vitro transposition reactions into E.coli cells by transformation (Table 1). Standard reaction conditions (10 nM donor) yielded a substantial fraction of recovered plasmid clones that exhibited the transposon marker (4.0% with supF-Mu and 11.8% with cat-Mu of clones carrying the target plasmid marker). From 1 µl of a standard reaction the total number of colonies recovered with the transposon marker was of the order of 103 when recipient cells of 106-107 c.f.u./µg DNA competence were used. Even when the donor DNA concentration was decreased to 0.1 nM, we recovered more than 100 colonies using electroporation and cells of 2 × 107 c.f.u./µg DNA competence (data not shown).
Table 1. Recovery of mini-Mu transposon insertions
| Donor (nM) | MuA | Total plasmidsa | Transposon selected for | Percentage | |
| Selection (µg/ml) | Cam (5) | Cam (5), Amp (25), Tet (8) | |||
| SupF-Mub | 10 | - | 2.1 × 105 | 0 | 0 |
| 10 | + | 1.2 × 105 | 4.8 × 103 | 4.0 | |
| 1 | + | 1.6 × 105 | 1.1 × 103 | 0.7 | |
| Selection (µg/ml) | Amp (100) | Amp (100), Cam (5) | |||
| Cat-Mub,c | 10 | - | 5.8 × 104 | 0 | 0 |
| 10 | + | 2.2 × 104 | 2.6 × 103 | 11.8 | |
| 1 | + | 2.4 × 104 | 2.1 × 102 | 0.9 |
bSupF-Mu was targeted into plasmid pSC SK+ and integrants selected on strain MC1061/P3. Cat-Mu was targeted into pUC19 and integrants selected on strain DH5[alpha]. Selections were performed on LB plates containing appropriate antibiotic(s). Cam, chloramphenicol; Amp, ampicillin; Tet, tetracycline.
cThe assembly of transpososomes is somewhat slower with the cat-Mu transposon compared to supF-Mu (data not shown). Therefore, a longer reaction time with cat-Mu (4 h instead of 1 h) was used to compensate for the difference.
To verify transposon integration events, 86 plasmid clones selected for supF-Mu insertions were subjected to a diagnostic restriction analysis with BamHI (data not shown). The transposon DNA was detected in all clones and, for 74 clones, the restriction digestion revealed a band pattern consistent with a single insertion of the transposon into the target. The rest of the clones appeared to have gained two or more insertions; based on the reaction product profile this was expected (Fig. 2A). We then determined the sequences flanking the transposon in 52 individual clones and thereby localized the integration sites (Fig. 3A). All the sequenced clones exhibited a 5 bp sequence repetition flanking the transposon. Thus, the in vitro transposition system used accurately recapitulated the genuine Mu transposition reaction with the hallmarkof a 5 bp target site duplication (31,32); the compiled insertion site data (not shown) is consistent with the consensus target site, NPyG/CPuN, determined earlier (33). Mini-transposons were distributed fairly evenly throughout the plasmid, except within the essential origin of replication (Fig. 3A; see below). No orientation dependence of transposon insertions into the target plasmid was observed.
The fine-scale distribution of integrated transposons was analyzed with supF-Mu insertions by a PCR-based assay (Fig. 3B). In the assay, the primary transposition reaction products are amplified using a pair of primers (one target-specific and one transposon-specific) to reveal integration sites in the sequence level. Most if not all phosphodiester bonds served as targets, but with different frequencies (lanes 3 and 6). Overall, the pattern was relatively even; no discernible region of DNA exhibited an exceptionally high or low frequency of integration. However, certain phosphodiester bonds were clearly represented more frequently than others. Thus, it appears that even though some particular sites are more favored than others as targets, the relative integration frequency into longer DNA fragments is expected to be fairly even (see below). This is in clear contrast to the in vitro Mu reaction in the presence of MuB protein. A regional target preference pattern was obtained, associated with DNA sites exhibiting a high affinity for MuB (33).
The influence of target DNA conformation on target site selection was then studied by the above fine-scale analysis with supF-Mu as donor and three different forms of plasmid pUC19 (supercoiled, open circular and linear) as targets. Identical distribution of integrated transposons was observed with each plasmid form (data not shown). In addition, quantification of the reaction components from a time course experiment with 33P-labeled supF-Mu donor DNA indicated that, with all three substrates, the reaction proceeded with similar kinetics (data not shown). These two experiments were then repeated with a 50 bp Mu R-end fragment as a donor and corresponding results were obtained (data not shown). Thus, all three forms of the plasmid DNA served as equal targets for the reaction. A comparison of the results showed, in addition, that the donor DNA, either the Mu R-end fragment or supF-Mu, had no influence on the target choice.
Since the generation of mutants by Mu transposition was highly efficient, we examined the use of the Mu system for functional analysis of DNA. The analysis was based on a comparison of two pools of transposon mutants: unselected and selected for a function. As a test case, we selected for plasmid replication functions and localized the DNA region of interest by two physical assays. In the first assay, the selected pool was subjected to restriction analysis (Fig. 4) and the amount of each transposon-containing restriction fragment was quantitated by Southern blotting and phosphorimaging. Certain restriction fragments were under-represented and they mapped to the plasmid origin of replication. The result thus indicated that functional mapping of a selectable gene region can be performed by first selecting a transposon mutant pool and then diagnosing the pool by a simple restriction analysis. In the second assay, the selected and unselected transposon mutant pools were PCR amplified using unlabeled transposon-specific and 33P-labeled target-specific primers (Fig. 5). The radioactive PCR products were then analyzed by denaturing polyacrylamide gel electrophoresis and visualized by autoradiography. When the obtained band patterns were compared between mutant pools, two types of outcome were observed. The primer pairs that yielded amplification products from DNA regions known not to be involved in plasmid DNA replication produced very similar patterns not depending on whether the pool was selected or not (lanes 1-4 and 9-14). On the other hand, those primer pairs that yielded amplification products from the origin of replication region showed a clear difference between the selected and unselected pools (lanes 5-8). In the reaction with the selected pool, a set of bands was missing, creating a clear `footprint'. The experiment thus demonstrated that a functional, high resolution genetic analysis can be performed efficiently using the Mu in vitro transposition system.
Figure 4. Localization of the functional DNA region by restriction analysis. (A) Restriction fragments generated by digestion of pUC19 with BfaI and AseI enzymes shown in a linear representation of the plasmid. (B) An EtBr-stained gel showing pUC19 (lanes 1 and 3) and the selected transposon mutant pool (lanes 2 and 4) digested with BfaI and AseI. The Tn after the numbers indicating fragment lengths denotes cat-Mu insertion. (C) A Southern blot (cat-Mu as a probe) of a gel similar to that in (B) but with a longer run time. The lanes and fragments are marked accordingly. See (D) for explanation of arrows. (D) Quantitation of the hybridization signal (arbitrary units) from Southern blots shown as a function of DNA fragment length. (Left) The unselected plasmid pool digested with BfaI as a control; the radioactivity detected in each restriction fragment correlates with the size of the fragment, showing that the distribution of the transposon insertions is relatively even. (Right) The selected plasmid pool digested with BfaI and AseI. Those fragments located at the plasmid origin of replication are clearly under-represented as judged by the amount of radioactivity (see arrows).
Figure 5. Genetic footprinting analysis of the selected plasmid pool. `Forward' primers (33P-labeled) hybridizing to different locations in plasmid pUC19 and `reverse' primer (unlabeled) hybridizing to mini-transposon DNA are shown. An autoradiogram of a 6% urea-polyacrylamide gel. On top are indicated the forward primers used in combination with the invariant reverse primer. The unselected pool (-) and selected pool (+) were used as templates as shown. Lane K is a PCR control reaction (pUC19 as template) in which all six forward primers were used with the reverse primer. Footprints seen in lanes 6 and 8 are marked with striped bars.
DISCUSSION
We developed a powerful methodology, based on the MuDNA transposition reaction, for genetic analysis. The described Mu in vitro reaction differs from the authentic Mu transposition reaction in vivo in that the two-step process of cleavage and strand transfer is reduced to the latter step only; this is facilitated by the use of donor DNA in a pre-cut configuration, which ensures efficient assembly and sufficient stability of Mu transpososomes (24,25). The reaction thus mimics `cut and paste' transposition (20,21) in which the transposon fragment is first physically liberated from the donor DNA. In addition, the accessory protein and DNA cofactors involved in Mu transposition (17,23) are not utilized.
Because the underlining Mu in vitro transposition reaction is highly efficient, the production of transposon mutants en masse is straightforward; under the reaction conditions used ~10% of target plasmids were recovered as integrants. Based on our results, if cells of very high competence (e.g. 109 c.f.u./µg DNA) were used, the maximum number of transposon-containing plasmids obtained would be of the order of 107-108 per µg DNA. Therefore, essentially any plasmid can be subjected to saturation mutagenesis. In addition, fairly large DNA molecules such as YACs, PACs and BACs are expected to be targets of efficient mutagenesis. Whenever mutagenized chromosomal DNA can be stably introduced into the genome of an organism by means of homologous recombination, possible for example with naturally transformable bacteria (15,16) and E.coli(34), genome-wide transposon mutagenesis projects are feasible (see for example 15,16) and will benefit from the Mu system. Saturation mutagenesis of targeted regions of microbial genomes can also be envisioned as well as massive mutagenesis projects involving the whole genome of yeast and possibly that of other organisms.
The accuracy of the Mu system is sufficient for demanding tasks. We have used the system for DNA sequencing purposes and analyzed more than 200 insertion sites, all with accurate target site duplication (27; data not shown). Such a high accuracy with relatively even distribution of integrations makes the Mu system an ideal choice for making libraries of mutant proteins, e.g. by scanning mutagenesis (14), for genetic footprinting (11,12) and other purposes. In principle, any DNA segment attached between Mu ends can be used as a mini-transposon which allows the construction of `design' transposons for different tasks. Especially valuable will be transposons aimed at protein engineering applications. These include additions, into the gene of interest, of sequences coding for e.g. a functional protein domain, identifiable sequence tag (such as a His-Tag or antibody epitope) or translation stop codon. In general, restructuring of DNA molecules by disseminating custom-made transposons into plasmids can be viewed as an alternative means, i.e. without restriction enzymes or PCR, to construct recombinant DNA molecules.
DNA in various conformations was equally effective as a target in the Mu mutagenesis system. The practical consequence is that versatile substrates including restriction fragments or linear virus genomes can be efficiently utilized as targets. Because of genome packaging constraints of viruses, a short transposon such as supF-Mu is expected to be least harmful to the virus and therefore most beneficial for mutagenesis purposes (for a conceptually similar in vivo approach see 35).
Classically, transposon mutants have been analyzed individually for function using selection or by screening for the phenotype; such an approach is applicable to the Mu insertion mutants as well. In each mutant, the transposon sequence provides a primer binding site and thus a direct access to the gene of interest through sequence analysis. We showed, in addition, that genetic analyses, utilizing pools of transposon mutants for functional mapping of important gene regions, are feasible with the Mu system. The restriction enzyme assay is easy to perform and indicates the region of interest with minimum effort. The PCR-based genetic footprinting strategy, however, extends the resolution to the nucleotide level.
The Mu system proved to perform optimally by all three criteria critical for efficient utilization of in vitro transposition technology: (i) efficiency; (ii) accuracy; (iii) even distribution of target sites. While some of the other in vitro DNA transposition systems currently available (9,12,15,16,36-46, and references therein) also seem to function fairly adequately in these respects, in particular those based on retroelement integration reactions and many of those based on wild-type reactions of bacterial transposons, appear to be compromised in one or more of the above three parameters. However, in some of the latter cases the performance of the system has been improved substantially by the use of a mutant version of a protein component in the reaction (15,44). In the near future, the currently existing in vitro transposition applications based on Mu and other mobile elements, some of which are already commercially available (9,15,27,44), are expected to yield a new set of routine methods for molecular biology. Finding novel uses for such reactions, especially in gene, genome and protein analyses as well as in transgenic studies, will be a challenge worth pursuing.
ACKNOWLEDGEMENTS
We thank Arja Lamberg, Alan Schulman and Dik van Gent for critical reading of the manuscript and Lars Paulin for his help in DNA sequence analysis. Financial support (to H.S.) was obtained from the Academy of Finland, Technology Development Center and Biocenter Helsinki.
REFERENCES
*To whom correspondence should be addressed. Tel: +358 9 7085 9516; Fax: +358 9 7085 9366; Email: harri.savilahti{at}helsinki.fi The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
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M. Pajunen, H. Turakainen, E. Poussu, J. Peranen, M. Vihinen, and H. Savilahti High-precision mapping of protein protein interfaces: an integrated genetic strategy combining en masse mutagenesis and DNA-level parallel analysis on a yeast two-hybrid platform Nucleic Acids Res., August 15, 2007; (2007) gkm563v1. [Abstract] [Full Text] [PDF]
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L. Orsini, M. Pajunen, I. Hanski, and H. Savilahti SNP discovery by mismatch-targeting of Mu transposition Nucleic Acids Res., March 19, 2007; 35(6): e44 - e44. [Abstract] [Full Text] [PDF]
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M. Varrelmann, E. Maiss, R. Pilot, and L. Palkovics Use of pentapeptide-insertion scanning mutagenesis for functional mapping of the plum pox virus helper component proteinase suppressor of gene silencing J. Gen. Virol., March 1, 2007; 88(3): 1005 - 1015. [Abstract] [Full Text] [PDF]
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 G. Donofrio, S. Cavirani, A. Vanderplasschen, L. Gillet, and C. F. Flammini Recombinant Bovine Herpesvirus 4 (BoHV-4) Expressing Glycoprotein D of BoHV-1 Is Immunogenic and Elicits Serum-Neutralizing Antibodies against BoHV-1 in a Rabbit Model Clin. Vaccine Immunol., November 1, 2006; 13(11): 1246 - 1254. [Abstract] [Full Text] [PDF]
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 M. Krupovic, H. Vilen, J. K. H. Bamford, H. M. Kivela, J.-M. Aalto, H. Savilahti, and D. H. Bamford Genome Characterization of Lipid-Containing Marine Bacteriophage PM2 by Transposon Insertion Mutagenesis. J. Virol., September 1, 2006; 80(18): 9270 - 9278. [Abstract] [Full Text] [PDF]
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A.-H. Saariaho and H. Savilahti Characteristics of MuA transposase-catalyzed processing of model transposon end DNA hairpin substrates Nucleic Acids Res., June 6, 2006; 34(10): 3139 - 3149. [Abstract] [Full Text] [PDF]
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J. H. Yu and D. V. Schaffer Selection of novel vesicular stomatitis virus glycoprotein variants from a Peptide insertion library for enhanced purification of retroviral and lentiviral vectors. J. Virol., April 1, 2006; 80(7): 3285 - 3292. [Abstract] [Full Text] [PDF]
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E. Poussu, J. Jantti, and H. Savilahti A gene truncation strategy generating N- and C-terminal deletion variants of proteins for functional studies: mapping of the Sec1p binding domain in yeast Mso1p by a Mu in vitro transposition-based approach Nucleic Acids Res., July 8, 2005; 33(12): e104 - e104. [Abstract] [Full Text] [PDF]
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D. D. Jones Triplet nucleotide removal at random positions in a target gene: the tolerance of TEM-1 {beta}-lactamase to an amino acid deletion Nucleic Acids Res., May 16, 2005; 33(9): e80 - e80. [Abstract] [Full Text] [PDF]
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 M. I. Pajunen, A. T. Pulliainen, J. Finne, and H. Savilahti Generation of transposon insertion mutant libraries for Gram-positive bacteria by electroporation of phage Mu DNA transposition complexes Microbiology, April 1, 2005; 151(4): 1209 - 1218. [Abstract] [Full Text] [PDF]
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 M. J. Song, S. Hwang, W. H. Wong, T.-T. Wu, S. Lee, H.-I Liao, and R. Sun Identification of viral genes essential for replication of murine {gamma}-herpesvirus 68 using signature-tagged mutagenesis PNAS, March 8, 2005; 102(10): 3805 - 3810. [Abstract] [Full Text] [PDF]
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 S. Kiljunen, H. Vilen, M. Pajunen, H. Savilahti, and M. Skurnik Nonessential Genes of Phage {phi}YeO3-12 Include Genes Involved in Adaptation to Growth on Yersinia enterocolitica Serotype O:3 J. Bacteriol., February 15, 2005; 187(4): 1405 - 1414. [Abstract] [Full Text] [PDF]
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C. Zhang, D. Kitsberg, H. Chy, Q. Zhou, and J. R. Morrison Transposon-mediated generation of targeting vectors for the production of gene knockouts Nucleic Acids Res., February 7, 2005; 33(3): e24 - e24. [Abstract] [Full Text] [PDF]
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 V. Petyuk, J. McDermott, M. Cook, and B. Sauer Functional Mapping of Cre Recombinase by Pentapeptide Insertional Mutagenesis J. Biol. Chem., August 27, 2004; 279(35): 37040 - 37048. [Abstract] [Full Text] [PDF]
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C. Lo, K. Adachi, J. R. Shuster, J. E. Hamer, and L. Hamer The bacterial transposon Tn7 causes premature polyadenylation of mRNA in eukaryotic organisms: TAGKO mutagenesis in filamentous fungi Nucleic Acids Res., August 15, 2003; 31(16): 4822 - 4827. [Abstract] [Full Text] [PDF]
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 I. Y. Goryshin, T. A. Naumann, J. Apodaca, and W. S. Reznikoff Chromosomal Deletion Formation System Based on Tn5 Double Transposition: Use For Making Minimal Genomes and Essential Gene Analysis Genome Res., April 1, 2003; 13(4): 644 - 653. [Abstract] [Full Text] [PDF]
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H. Vilen, J.-M. Aalto, A. Kassinen, L. Paulin, and H. Savilahti A Direct Transposon Insertion Tool for Modification and Functional Analysis of Viral Genomes J. Virol., December 6, 2002; 77(1): 123 - 134. [Abstract] [Full Text] [PDF]
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T. A. Naumann, I. Y. Goryshin, and W. S. Reznikoff Production of combinatorial libraries of fused genes by sequential transposition reactions Nucleic Acids Res., November 1, 2002; 30(21): e119 - e119. [Abstract] [Full Text] [PDF]
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Y. S. N. Butterfield, M. A. Marra, J. K. Asano, S. Y. Chan, R. Guin, M. I. Krzywinski, S. S. Lee, K. W. K. MacDonald, C. A. Mathewson, T. E. Olson, et al. An efficient strategy for large-scale high-throughput transposon-mediated sequencing of cDNA clones Nucleic Acids Res., June 1, 2002; 30(11): 2460 - 2468. [Abstract] [Full Text] [PDF]
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A. Lamberg, S. Nieminen, M. Qiao, and H. Savilahti Efficient Insertion Mutagenesis Strategy for Bacterial Genomes Involving Electroporation of In Vitro-Assembled DNA Transposition Complexes of Bacteriophage Mu Appl. Envir. Microbiol., February 1, 2002; 68(2): 705 - 712. [Abstract] [Full Text] [PDF]
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V. I. Zabarovska, R. Z. Gizatullin, A. N. Al-Amin, R. Podowski, A. I. Protopopov, S. Lofdahl, C. Wahlestedt, G. Winberg, V. I. Kashuba, I. Ernberg, et al. A new approach to genome mapping and sequencing: slalom libraries Nucleic Acids Res., January 15, 2002; 30(2): e6 - e6. [Abstract] [Full Text] [PDF]
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F. H. E. Schagen, H. J. Rademaker, S. J. Cramer, H. van Ormondt, A. J. van der Eb, P. van de Putte, and R. C. Hoeben Towards integrating vectors for gene therapy: expression of functional bacteriophage MuA and MuB proteins in mammalian cells Nucleic Acids Res., December 1, 2000; 28(23): e104 - e104. [Abstract] [Full Text] [PDF]
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M. C. Biery, F. J. Stewart, A. E. Stellwagen, E. A. Raleigh, and N. L. Craig A simple in vitro Tn7-based transposition system with low target site selectivity for genome and gene analysis Nucleic Acids Res., March 1, 2000; 28(5): 1067 - 1077. [Abstract] [Full Text] [PDF]
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S. Haapa-Paananen, H. Rita, and H. Savilahti DNA Transposition of Bacteriophage Mu. A QUANTITATIVE ANALYSIS OF TARGET SITE SELECTION IN VITRO J. Biol. Chem., January 18, 2002; 277(4): 2843 - 2851. [Abstract] [Full Text] [PDF]
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L. Hamer, K. Adachi, M. V. Montenegro-Chamorro, M. M. Tanzer, S. K. Mahanty, C. Lo, R. W. Tarpey, A. R. Skalchunes, R. W. Heiniger, S. A. Frank, et al. Gene discovery and gene function assignment in filamentous fungi PNAS, April 24, 2001; 98(9): 5110 - 5115. [Abstract] [Full Text] [PDF]
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A. M. Gehring, J. R. Nodwell, S. M. Beverley, and R. Losick Genomewide insertional mutagenesis in Streptomyces coelicolor reveals additional genes involved in morphological differentiation PNAS, August 15, 2000; 97(17): 9642 - 9647. [Abstract] [Full Text] [PDF]
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T. Kekarainen, H. Savilahti, and J. P.T. Valkonen Functional Genomics on Potato Virus A: Virus Genome-Wide Map of Sites Essential for Virus Propagation Genome Res., April 1, 2002; 12(4): 584 - 594. [Abstract] [Full Text] [PDF]
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