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Figure 1 Proposed IS911 transposition pathway. Transposon DNA (bold lines), donor backbone sequences (fine lines), target DNA (dotted lines), IRL and IRR (small circles). The different steps of IS911 transposition are shown: synapsis of the ends in a plasmid donor to generate synaptic complex A; strand cleavage at one end (the donor end) and transfer to the other (the target end) to form a figure-of-eight structure in a reaction that requires only the transposase OrfAB; second strand resolution requiring host functions but no IS911 proteins to generate the transposon circle; synapsis with a target DNA molecule (synaptic complex B); cleavage of the IRR−IRL junction; and transfer into the target, which requires the transposase OrfAB. Final insertion requires OrfAB and is greatly stimulated by OrfA.
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 | Figure 2 Genetic organization and structure−function map of OrfAB and OrfA. Top: cartoon of IS911. Dark grey box, left terminal inverted repeat (IRL); triangle, right terminal inverted repeat (IRR); white boxes, reading frames orfA and orfB; 0 and -1, relative reading phases; pIRL, endogenous promoter; vertical line, point of translational frameshifting. Bottom: organization of IS911 proteins together with their molecular mass: black square,  -helix−turn−-helix motif (HTH); ellipses, individual heptads of leucine zipper (LZ); cross-hatched box, catalytic domain; black vertical lines, DDE signature. Programmed translational frameshifting that fuses orfA and orfB to generate the transposase OrfAB occurs within the fourth heptad. The LZ of OrfA and OrfAB therefore differ in their fourth heptad.
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Figure 3 Targeted insertion next to a single IR in vivo: influence of the IS911 proteins. (A) Experimental scheme. Top panel: circle formation from the donor plasmid. The general structure of the donor plasmids is indicated. They are derivatives of p15A and contain an artificial transposon composed of 52 bp of the left and 52 bp of the right IS911 end (dark grey boxes) flanking a gene for resistance to chloramphenicol (Cm; light grey box). The individual plasmids are described in more detail in (B). The cartoon shows the formation of transposon circle intermediates, which are subsequently inserted into the target plasmid (bottom panel). Bottom panel: structure of the target plasmid and the product of targeted insertion. Plasmid pBST1 carries a mutated IRL sequence (grey box) with the indigenous promoter pIRL and a gene fusion between the 5' part of orfA and lacZ from the eighth codon (orfA−lacZ) (cross hatched box), a pBR322 origin of replication (unfilled oval) and the  -lactamase (bla) gene. The IR-targeted product that would result from the insertion of the CmR transposon 3 bp from the resident IRL* reconstitutes the strong pjunc promoter and activates the orfA−lacZ gene. The position and orientation of the two promoters are indicated by an arrow. This type of targeted IRL* insertion is observed, after transformation, on MacConkey lactose plates. (B) Structure of the donor plasmids, transposition frequency and percentage of lac+ colonies. The left column presents the different plasmids used to supply IS911 proteins and the artificial IS911 transposon. pCL19 carries the wild-type configuration of IS911 orfs, which drives expression of both OrfA and OrfAB proteins under the control of the lacUV5 promoter (plac). pCL21 and pCL22 carry the orfAB gene alone, where the A6G frameshift signal was exchanged for CA5G (Polard et al., 1992) to artificially fuse the orfA and orfB reading frames under the control of placUV5. It also carries the E.coli paraBAD and the araC gene, which encodes the arabinose-inducible repressor. In addition, pCL22 carries the orfA gene, which drives expression of OrfA alone (orfA), under the control of paraBAD. The direction of gene transcription is indicated by curved arrows, and the position and orientation of the promoter are indicated by an arrow. The small open circles correspond to transcription terminators of the E.coli rrnB operon, and the small white boxes represent phage T4 transcription and translation termination signals flanking the streptomycin/spectinomycin resistance cassette (SpSm). lacIq, the lacIq allele. The two right-hand columns indicate the transposition frequency (Transptn;  106) and the percentage of lac+ (% lac+) colonies obtained on MacConkey lactose 1% Cm in the absence (-) or presence (+) of 1 mM IPTG, and in the absence (-) or presence (+) of 1% arabinose. The transposition frequency is expressed as the ratio of CmR transformants compared with ApR transformants and the percentage of lac+ is expressed as the number of CmR lac+ transformants as a percentage of total transformants (CmR).
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 | Figure 4 Targeted insertion into a two-ended IS911 derivative in vitro. (A) Structure of the substrate and target, and a targeted insertion product. The cartoons show the transposon circle employed as substrate and pAPT182 employed as target plasmid together with an IRL*- targeted insertion product. The symbols are identical to those presented in Figure 3. The insertion reaction included OrfAB at 0.42  M. (B) Junction structure of insertion products. The direction of gene (Cm and orfA−lacZ) transcription is indicated by arrows. The number of clones for each type of junction structure obtained is indicated. (C) Variation in junction spacing. Although the majority of insertions occur at a distance of 3 bp from the resident target IR, less frequent spacings of 2 and 4 bp are observed. The cartoons show how these two types of IR integration may be generated. The vertical arrows show the position of nucleophilic attack on the target plasmid. The black circles correspond to the 3 bp duplicated in the target plasmid.
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Figure 5 Targeted insertion into a single IR derivative in vitro. (A) Efficiency of transposition using a target plasmid carrying mutated copies of IRR and IRL. The figure shows a physical map of the target plasmid. These were constructed from a pBR322-based plasmid and all included a selectable ampicillin resistance gene (bla). The right and left terminal inverted repeats, IRL and IRR, are represented by a grey box and a grey pointed box, respectively. Plasmids pCL12 and pCL14 carry IRR* and IRL*, in which the 5'-CA terminal dinucleotide has been mutated to 5'-TC, inverted with respect to each other. Both IRs were cloned at the same position in the plasmid. The parent plasmid pCL15 does not carry an IR. Insertion reactions were carried out with an OrfAB concentration of 0.32 M and the products were introduced into JS238 by electroporation. The number of CmR colonies obtained in parallel is shown below for each target plasmid. These values are from a single experiment, although similar values were obtained in successive experiments. They are a measure of the relative efficiency of insertion of the CmR transposon circle. (B) Junction structure of targeted insertion products. The figure shows the configuration of IRs at the newly formed junction with pCL12 obtained from sequencing. bla and Cm indicate the plasmid- and transposon circle-associated antibiotic resistance genes, and the arrows show their direction of transcription. (C) Competition experiments using two target plasmids. pCL11 and pCL16 were constructed from a pBR322-based plasmid that included the selectable tetracycline resistance gene (Tc). pCL12 [see (A)] and pCL11 carry IRR* cloned in the same position and orientation. pCL15 [see (A)] and pCL16 are IR less. Insertion reactions were carried out with an OrfAB concentration of 0.32 M and the products were introduced into DH5 by transformation. The values below the plasmids represent the absolute number of CmR colonies from one experiment that were also ApR or TcR. They represent the relative target efficiency of the two plasmids present in the reaction. (D) Efficiency of transposition using a target plasmid carrying wild-type copies of IRR and IRL. pCL24 and pCL26 each carry a wild-type IRL copy inverted with respect to each other, and pCL25 and pCL27 each carry an IRR inverted with respect to each other. The IRs were cloned at the same position in the plasmid as those in pCL12 and pCL14. Insertion reactions were carried out under the same conditions and in parallel with pCL12 and pCL14 [see (A)].
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 | Figure 6 Targeted insertion into a two-ended IS911 derivative in vitro as a function of OrfAB and OrfA. (A) Efficiency of targeted and non- targeted insertion as a function of the OrfAB/OrfA ratio. The efficiency of targeted and non-targeted insertion was measured as the number of CmR lac+ and CmR lac- colonies, respectively, following transformation of JS238 with the reaction products. The concentration of OrfAB was kept constant (0.42 M) and the concentration of OrfA was varied. The concentration of OrfA used was 0.0, 0.7, 1.4, 2.8, 4.2, 5.6, 7 and 8.4 M, corresponding to the molar ratios of OrfAB/OrfA 1/0, 1/1.8, 1/3.6, 1/7, 1/11, 1/14, 1/18 and 1/21. (B) Percentage of IRL*-targeted insertion as a function of the OrfAB/OrfA ratio. The percentage of IRL*-targeted insertion is expressed as the number of CmR lac+ transformants as a percentage of total (CmR) transformants. A similar experiment performed with proteins obtained from an overexpressed wild-type (WT) configuration of IS911 orfs (0.6 g) was also included.
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Figure 7 Non-IR-targeted insertions. (A) Distribution of insertion sites in pAPT182. Genetic elements are indicated in the inner circles and include the following: gene encoding ampicillin resistance (bla), the ends IRR* and IRL*, the pBR322 origin of replication (Ori) and the reporter gene lacZ (see description of pAPT182, Figure 4A). The lines in the inner circle correspond to one orientation of insertion and those in the outer circle correspond to the opposite orientation. The numbers indicate the total number of insertion at a given site. The 'funnels' indicate IR-targeted insertions and numbers present the number of insertions found at a specific site. The left section shows the results obtained with OrfAB alone (0.42 M), while the right section shows those obtained in the presence of both OrfAB (0.42 M) and OrfA (0.7 and 7 M). (B) Alignment of target sites used by IS911. Forty-four target sites and the flanking DNA sequences were aligned as 88 half-sites. The analysis was performed on a 20 bp window flanking the duplicated insertion site. A table was generated by compiling the sum of each nucleotide at each position in a 20 4 table. The significance of the nucleotide distribution at a given site was determined by the  2 Pearson test [2 =  (o - c)2/c], where o and c are the observed and calculated values, respectively. It took into consideration the overall nucleotide composition of the target plasmid pAPT182 (22.59% A, 23% T, 27.28% G and 27.11% C) as calculated values. The positions at which a nucleotide bias occurs with >99% (P < 0.01) confidence levels are indicated by a '+'.
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 | Figure 8 A proposed mechanism for IR-targeted insertion. Transposon circle DNA is shown (bold lines), as is target DNA (dotted lines). The small white circles represent IRL and IRR, and the small grey circles represent IRL or IRR carrying the terminal 5'-CA to 5'-TC mutation. Insertion next to the resident mutated IR in the target plasmid is proposed to initiate by the formation of an intermolecular OrfAB-mediated synaptic complex that resembles the intramolecular synaptic complex A (Figure 1). A single-strand cleavage of one end of the transposon circle and site-specific transfer to the target plasmid would create an intermolecular single-strand bridge. Simple cleavage at the second transposon end and intermolecular strand transfer would result in simple insertion of the transposon circle.
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