EP1590460A1 - Hyperactive, non-phosphorylated, mutant transposases of mariner mobile genetic elements - Google Patents

Abstract

The invention relates to hyperactive, non-phosphorylated, mutant transposases of mariner mobile genetic elements. The invention also relates to recombinant nucleotide sequences encoding such transposases. The invention further relates to a method of producing said transposases and to the use thereof for in vitro or in vivo transposition.

Description

 TRANSPOSASES OF MOBILE GENETICS

MUTANT, NON-PHOSPHORYLABLE AND HYPERACTIVE

The present invention relates to the field of molecular biology relating to transposable elements. The invention relates more particularly to the improvement of the properties of natural transposases of mobile mariner genetic elements, for the purposes of their use in biotechnologies.

The subject of the present invention is mutant, non-phosphorylatable, hyperactive transposases of mobile mariner genetic elements.

The invention also relates to recombinant nucleotide sequences encoding such transposases.

In addition, the invention relates to a process for the production of these transposases, as well as the use of the latter for transposition In vitro or in vivo.

Transposable elements (TE) or mobile genetic elements (EGM) are small DNA fragments capable of moving from one chromosomal site to another (Renault et al., 1997). These DNA fragments are characterized by inverted repeat (ITR) sequences located at the 5 ′ and 3 ′ positions distally. An enzyme encoded by the TEs themselves, transposase, catalyzes the transposition process of the latter.

TEs have been identified in both prokaryotes and eukaryotes (see a reference work in this area: Craig et al., 2002).

TEs are divided into two classes according to their transposition mechanism. On the one hand, class I elements, or retrotransposons, transpose via the reverse transcription of an RNA intermediate. On the other hand, class II elements transpose directly from a site chromosome to another, via a DNA intermediary, according to a “cut and paste” type mechanism.

In prokaryotes, a large number of TEs have been identified to date. Mention may be made, for example, of insertion sequences such as IS, and of transposons, such as Tn5.

In eukaryotes, class II elements include five families: P, PiggyBac, hAT, helitron and Tel-mariner.

The mobile mariner genetic elements (or MLE for “mariners-like elements”) constitute a large group of TE class II, belonging to the Tel-mariner superfamily (Plasterk et al., 1999).

The ability of TE transposases to mobilize more or less long DNA fragments, homologous or heterologous, comprising sequences of interest, to insert them into target nucleic acids, in particular in the chromosome of a host, has been and is still widely used in the field of biotechnology, especially in the field of genetic engineering.

Among TEs, MLEs have particularly advantageous properties for use in biotechnologies, in particular in genetic engineering and functional genomics. The following properties may for example be mentioned without limitation:

(i) MLE are small transposons, easy to handle.

(ii) The mechanism for transposing MLE is simple. Indeed, the transposase is capable of catalyzing, on its own, all the stages involved in the process of transposition of MLE. It is moreover necessary and sufficient to ensure the mobility of MLE in the absence of host factors (Lampe et al., 1996).

(iii) MLE is characterized by a very wide ubiquity in prokaryotes and eukaryotes. The first MLE, Dmmarl, also called Mos-1, was discovered at Drosophila Mauritiana by Jacobson and Hartl (1985). Subsequently, many related elements have been identified in genomes belonging in particular to protozoa, fungi, plants, invertebrates, cold-blooded vertebrates and mammals.

(iv) The transposition activity of MLE is highly specific, and does not stimulate mechanisms of

"Resistance" of the host genome, such as methylation interference phenomena [MIP; Jeong ef al. (2002); Martienssen and Colot (2001)] or via NRAs

[RNAi; Ketting et al. (1999); Tabara et al. (1999)]. Transposition events can be controlled by various factors, such as temperature and pH.

Consequently, the potential applications of MLE in biotechnology, in particular as tools for genetic recombination, are considerable.

However, the transposition efficiency of natural MLE, compared to that of other TE, remains low. In particular, MLEs appear to be less active in eukaryotes than other class II EGMs. Ultimately, the practical interest of natural MLE has so far been limited, since it is important, for the industrialist or the researcher, to have effective transposases, so as to reduce the number of manipulations, the cost and the time required to carry out the desired transpositions. The present invention precisely makes it possible to satisfy these requirements, by taking advantage of the advantages of MLE transposases, while overcoming their drawbacks.

The present invention highlights the fact that MLE transposases are phosphorylated in eukaryotes, by means of post-translational modifications (see Experimental Part below).

These post-translational modifications have two effects: on the one hand, they reduce the affinity of transposases for DNA; and on the other hand, they limit the conformational activation of these enzymes. This ultimately results in a reduced ability of phosphorylated transposases to catalyze transposition in eukaryotes. Also, in order to improve the transpositional properties of MLE transposases, the invention aims to suppress, by point mutagenesis, one or more phosphorylation sites of these enzymes.

According to a first aspect, the subject of the present invention is a mutant and hyperactive MLE transposase. According to a first embodiment, such a transposase has at least one mutation by conservative substitution of at least one phosphorylatable residue in at least one phosphorylation site, said conservative substitution rendering said site non-phosphorylable in vivo.

A mutant and hyperactive transposase according to the invention is therefore at least partially non-phosphorylable. In the following, such a transposase will be designated as being “mutant, non-phosphorylable and hyperactive”.

The term “hyperactive” transposase is understood here to mean a transposase whose transposition efficiency in eukaryotes is increased by a factor greater than or equal to approximately 10, preferably greater than or equal to approximately 25, more preferably greater than or equal to about 50, more preferably greater than or equal to about 100.

In particular, a transposase according to the invention is “hyperactive” for at least one function among: specific and non-specific binding to DNA, dimerization, transfer of DNA strands, and endonuclease and internalization properties nuclear (see Figure 2 below).

Thus, in the present context, the expression “hyperactivity of a transposase” means that the biological properties (or functions or activities) of this transposase are improved or increased. Each of the above-mentioned activities is necessary, but can be limiting, during the implementation of the transposition process. In in particular, each of these activities is capable of being regulated by post-translational phosphorylations in eukaryotes.

"DNA binding" involves three mechanisms of DNA recognition by MLE transposases. Transposases can bind DNA via their N-terminal domain, either non-specifically (first mechanism) or specifically via ITRs (second mechanism). These two link modes depend on the conformational state of the N-terminal domain. When the transposases are trapped in the synaptic complex, just after excision of the transposon (this complex comprising at least one transposase dimer linked by ITRs to the excised transposon), they can bind to DNA according to a third mechanism, for recognize the site of insertion into the target DNA.

MLE transposases are able to self-associate (“dimerization” properties in the context of the invention) according to two processes. They can dimerize (homodimers) when they are specifically linked to ITRs. They are also capable of oligomerizing (homodimers or homooligomers) when they are in an inactive and / or high concentration conformation, when they are not linked to ITRs. In the Mos-1 transposase, the region comprising the position T88 (see below) has been proposed as being involved in dimerization (personal data; Zhang et al., 2001).

The activity of "DNA strand transfer" is defined in the literature as covering the capacity of MLE transposases, which are phosphoryl transferases, to bind DNA fragments, concomitantly with the cutting of these.

The endonuclease properties are therefore very closely linked to the above-mentioned “DNA strand transfer” activity. The cleavage activities presented by MLE transposases are of three types: cleavage at the ends of the transposon during excision, linearization circular excision intermediates and cleavage of the site of insertion into the target DNA.

Finally, MLE transposases have two potential bipartite sites allowing nuclear internalization. The terms and expressions "activity", "function", "property",

"Biological activity", "biological function" and "biological property" are equivalent and respond to the usual meaning in the technical field of the invention. In the context of the invention, the activity in question is the enzymatic activity of a transposase (“transposase activity” or “transposase function”). Depending on the context, the expression “transposase activity” may generically designate all of the enzymatic activities of an MLE transposase, as mentioned above, or one or more of these activities. In any case, the meaning to be given to this expression will be deduced in a clear and unambiguous manner from the context by those skilled in the art.

For the purposes of the invention, a “mutation” designates a substitution of one or more bases in a nucleotide sequence, or of one or more amino acids in a protein sequence. More particularly, a “mutation” should here be understood as designating a substitution of at least one base of a codon of a nucleotide sequence encoding an MLE transposase, said substitution resulting, during the translation of the nucleotide sequence in question , the incorporation of a different (non-phosphorylatable) amino acid in place of the native amino acid (phosphorylatable), in the resulting protein sequence.

A mutation by substitution according to the invention must be “conservative”, ie, it must be a non-random substitution preserving and, preferably, improving one or more enzymatic activities of wild MLE transposases. Preferably, a “conservative” substitution in accordance with the invention improves all of the enzymatic activities of the MLE transposases. This is possible especially when the overall tertiary structure of the MLE transposase is conserved despite the substitution mutation (s) introduced. For the purpose of conserving the tertiary structure of a protein (here, of a transposase), and improving its biological activity, a person skilled in the art knows, thanks to his general knowledge, which amino acid substitutions may be appropriate and which substitutions must be avoided (Mornon et al., 2002).

In particular, those skilled in the art can refer to the following Table 1, which provides a non-limiting list of possible conservative substitutions.

Table 1

Wild residue Conservative substitution

Ala (A) Gly; Ser

Arg (R) Lys

Asn (N) Gin; His

Cys (C) Ser neutral amino acids

Gin (Q) Asn

Glu (E) Asp

Gly (G) Ala; Pro

His (H) Asn; Gin

Ile (1) Leu; Val

Leu (L) Ile; Val

Lys (K) Arg; Gin; Glue

Met (M) Leu; Tyr, Island

Phe (F) Met; Leu; Tire

Ser (S) Thr

Thr (T) Ser

Trp (W) Tyr

Tyr (Y) Trp; Phe

Val (V) Ile; Leu

In the case of mutagenesis carried out on phosphorylation sites, the substitutions may be wholly or partly conservative of the activities of the protein if these are for example performed as follows: substitution of a serine or a threonine with, in order of preference, a cysteine, an alanine, a valine, a leucine, a tyrosine or an arginine. A conservative substitution appropriate to the present context can in no case be carried out using, as a substitution residue, in particular a serine, a threonine, an aspartic acid, a glutamic acid or a proline.

Optionally, other suitable substitutions can be determined empirically or from the conservative substitutions exemplified in Table 1 above. In the context of the invention, the “phosphorylation sites” can in particular be sites of phosphorylation by the protein kinase AMPc, gMPc dependent, or by protein kinase C, or also by casein kinase 11. It can furthermore s 'act of putative phosphorylation sites, corresponding to consensus patterns known to those skilled in the art (PROSITE motif banks: httD: // ww.infobioαen.fr) as shown in Figure 1 below. The short sites, absent from PROSITE, and corresponding to 4 QS, QT, SQ and TQ dipeptides phosphorylatable by ATM kinases are also taken into account as being “putative phosphorylation sites” (Kastan et al., 2000; Kastan et al ., 2001).

In addition, the phosphorylation sites targeted by the invention can be conserved through at least two MLE transposases. In this case, they can be demonstrated by aligning the protein sequences of at least two MLE transposases. One of these sequences can be, for example, that of the Mos-1 transposase (see the alignment of the MLE transposase sequences deposited with the EMBL database under the access number DS36877). The residues of two distinct protein sequences thus aligned are called here "corresponding residues". In order to facilitate the identification of phosphorylatable residues capable of being mutated in the sequence of an EGM mariner transposase distinct from the sequence SEQ ID No. 2 (sequence of the Mos-1 transposase), these residues are identified, in the context of the present invention, as being “corresponding residues of the sequence of said EGM mariner transposase aligned with the sequence SEQ ID No. 2”. Thus, the phosphorylatable residues capable of being mutated by conservative substitution are chosen from:

- The following residues of the sequence SEQ ID No. 2: T11; T24; S28; T42; T88; S99; S104; T135; S147; T154; S170; T181; S200; T216; T255; and S305; or - the corresponding residues of the sequence of a mariner EGM transposase aligned with said sequence SEQ ID No. 2, said corresponding residues being chosen from:

- a serine; and

- a threonine. According to a second embodiment, a transposase according to the invention exhibits at least one mutation by conservative substitution of at least one phosphorylatable residue in each phosphorylation site, said conservative substitution making each site non-phosphorylable in vivo. According to a third embodiment, a transposase which is the subject of the present invention is such that said phosphorylatable residue which is the target of the mutation by conservative substitution is substituted by a non-phosphorylatable residue.

According to a fourth embodiment, a mutant, non-phosphorylatable, hyperactive transposase which is the subject of the invention comes from an MLE belonging to the Mauiana subfamily.

The “MLE” targeted in the context of the present invention have between them sequence homologies of at least 50%, preferably at least 75%, more preferably at least 80%, or more preferably still , at least 90%. Alternatively, the MLEs according to the invention have sequence homologies between them at least 80%, preferably at least 90%, more preferably at least 95%, or more preferably still, at least 98%. Thus, the MLEs in question have, over their entire length, at least 50%, preferably at least 75%, more preferably at least 80%, or even more preferably at least 90% of identical bases. Alternatively, the MLEs have, over their entire length, at least 80%, preferably at least 90%, more preferably at least 95% or, more preferably still, at least 98%, of identical bases. Identical bases can be consecutive, in whole or in part only. The MLEs thus envisaged can be of the same length, or of a different length if their sequences have, relative to one another, deletions or insertions of one or more bases.

The "mauritiana subfamily" gathers the MLE whose transposases are coded by sequences presenting, over their entire length or at the level of the regions coding for the N- and C-terminal domains only, a sufficient level of homology, that is that is to say at least 75%, to be included in the same line as the four sequences below, during phylogenetic studies carried out using the methods of parsimony and "Neighbor-Joining" on a dataset of 1000 subsamples (1000 "bootstrap") [See Felsenstein (1993) and Augé-Gouillou et al. (2000)]:

- Mos-1 transposase (SEQ ID No. 2, EMBL access number: X78906);

- Mdmar-1 tranposase from Mayetiola destructor (EMBL access number: U24436);

- Btmar-1 transposase from Bombus terrestris (sequence No. 3 in the alignment EMBL access number: DS36877); and

- Momar-1 transposase from Metaseuilius occidentalis (EMBL access number: U12279). According to a fifth embodiment, a mutant, non-phosphorylatable, hyperactive transposase which is the subject of the invention comes from MLE mos-1.

In particular, in a mutant, non-phosphorylatable, overactive transposase originating from MLE mos-1, one or more phosphorylatable residues from the following residues (with reference to the sequence SEQ ID No. 2): T11; T24; S28; T42; T88; S99; S104; T135; S147; T154; S170; T181; S200; T216; T255; and S305, are substituted with non-phosphorylatable residues. Advantageously, at least the phosphorylatable residue T88 or, alternatively, at least the phosphorylatable residue S104 of such a transposase is substituted by a non-phosphorylatable residue. In particular, at least the residues T88 and S104 are substituted by non-phosphorylatable residues. According to a second aspect, the present invention relates to a recombinant nucleotide sequence encoding a transposase as described above.

A “nucleotide sequence” or a “nucleic acid” according to the invention conforms to the usual meaning in the field of biology. These two expressions indifferently cover DNA and RNA, the former being for example genomic, plasmid, recombinant, complementary (cDNA), and the latter, messenger (mRNA), ribosomal (rRNA), transfer (tRNA). Preferably, the nucleotide and nucleic acid sequences of the invention are DNA.

According to a third aspect, the present invention relates to a recombinant vector comprising at least one recombinant nucleotide sequence encoding a transposase according to the invention.

Advantageously, such a recombinant vector allows the expression of said recombinant nucleotide sequence. According to a fourth aspect, the subject of the present invention is a recombinant host cell hosting at least one recombinant vector as described above.

Insofar as the MLEs have, to date, no host restriction, a recombinant host cell according to the invention can be of both eukaryotic and prokaryotic origin.

It can, for example and without limitation, be a eukaryotic cell coming from a yeast, a fungus, a plant, an insect (eg, Drosophila), a rodent or a mammal (eg, rabbit).

Alternatively, a cell suitable for implementing the invention can come from a bacterium (e.g., Escherichia coli).

According to a fifth aspect, the present invention relates to a process for the production of a mutant, non-phosphorylatable, hyperactive transposase, as described above.

According to a first embodiment, a method according to the invention comprises at least: a) cloning of the recombinant nucleotide sequence coding for the mutant, non-phosphorylatable, hyperactive transposase in an expression vector; b) transforming a host cell with said recombinant expression vector; and c) expression of said nucleotide sequence by said recombinant host cell. All of the steps implemented in the context of such a process use conventional techniques known to those skilled in the art (see for example, Sambrook and Russel, 2001).

In particular, the term "transformation" is here given a generic meaning in that it also covers, in addition to transformation in the strict sense, transduction by a viral vector and transfection, as many molecular biology techniques perfectly usual for those skilled in the art.

According to a second embodiment, such a method further comprises a prior step of obtaining said recombinant nucleotide sequence, by substitution, in the nucleotide sequence encoding the corresponding native transposase, of at least one nucleotide of a triplet or codon encoding a phosphorylatable residue, by another nucleotide, so that the resulting triplet encodes a non-phosphorylatable residue. This preliminary step involves directed mutagenesis techniques, known to those skilled in the art (Ausubel et al., 1994).

According to a third embodiment of the method which is the subject of the invention, a subsequent step of purification of said transposase is carried out. Preferably, within the meaning of the present invention, the abovementioned purification step consists in purifying, by conventional methods customary for those skilled in the art, the protein fraction having the desired enzymatic activity, and not the enzyme itself. even. It is precisely this active protein fraction thus purified that is called here "pure enzyme" or "pure transposase". According to this definition, at the end of this step, the presence of contaminating substances, including that of other proteins, in a minority amount is not excluded, as long as the transposase activity of interest is preserved, and that only this activity is detected. The detection of the enzymatic activity of interest can be carried out by conventional methods known to those skilled in the art (Ausubel et al., 1994).

During the implementation of a process as mentioned above, the host cell is chosen from prokaryotic cells, for example from bacteria, and eukaryotic cells, for example from yeasts, fungi, plants, insects and mammals. According to a sixth aspect, the present invention relates to uses of at least one mutant, non-phosphorylatable, hyperactive transposase, as described above, in the field of biotechnology.

According to a first embodiment, such a transposase is used for the in vitro transposition of a transposable DNA sequence of interest into a target DNA sequence.

According to a second embodiment, a use in accordance with the invention is carried out for the purposes of the in vivo transposition of a transposable DNA sequence of interest into the genome of the host. Alternatively, a transposase according to the invention may be useful for preparing a medicament intended to allow the transposition in vivo of a transposable DNA sequence of interest into the genome of the host.

The implementation of an in vitro or in vivo transposition falls within the general knowledge of a person skilled in the art in the field of the invention (Ausubel et al., 1994; Craig et al., 2002).

A “host” is understood here as being able to be an organism, eukaryotic or prokaryotic, or a tissue of an organism, or even a cell of an organism or a tissue.

The following figures are provided for illustrative purposes only and do not limit the subject of the present invention in any way.

Figure 1: Nucleotide sequence of the mos-1 transposon (SEQ ID No. 1) and protein sequence of the Mos-1 transposase (SEQ ID No. 2). The putative phosphorylation sites are located as follows:

- simple framework: putative phosphorylation site by protein kinase AMPc, gMPc dependent;

- dotted frame: putative site of phosphorylation by protein kinase C;

- double frame: putative site of phosphorylation by casein kinase II. The residues represented in clear in the sequence (QTQ; positions 87 to

89 of the protein sequence SEQ ID No. 2) correspond to the putative site of phosphorylation by the family of ATM kinases.

The circled residues are the Âsp residues involved in the catalytic triad characteristic of MLE transposases (D, D34-35 [D / E]).

Figure 2: Diagram representing the structure of the transposase of the Mos-1 element.

N-term: N-terminal domain responsible for linking to ITRs;

C-term: C-terminal domain responsible for the catalysis of DNA strand transfer; NLS: putative nuclear localization signal (or nuclear internationalization); HTH: propeller-turn-propeller motif; aa: amino acid.

The numbers indicate the positions of the amino acids.

The characteristic catalytic triad [D, D34 (D / E)] is reported. Figure 3: Schematic representation of the transposition test.

A): bacteria co-transformed with the expression vector encoding the transposase (Tnp) and the reporter vector for the transposition.

B): Transposition event after induction of the expression vector.

C): Determination of the transposition frequency. Figure 4: “Dots” of pBadR-Tnp mRNA hybridized with:

- the L27 probe (A); and

- the Tnp probe (B).

Figure 5: Graph showing the Tnp / Transposase mRNA correlation as a function of the culture conditions in the pBadR expression system. Figure 6: “Dots” of hybrid pKK-Tnp mRNA with:

- the L27 probe (A); and

- the Tnp probe (B).

Figure 7: Graph showing the Tnp / Transposase mRNA correlation as a function of the culture conditions in the pKK expression system. Figure 8: Photograph of a 10% SDS-PAGE protein gel of protein extracts (A) and autoradiogram of a delay on gel of transposases in the presence of 3 'ITR (B).

Track 1: pBadR control Tracks 2 to 7: Tnp from the pBadR-Tnp system (2: 1% glucose, 3: control, 4: 0.001% ara, 5: 0.01% ara, 6: 0.1% ara, 7: 1% ara)

Lane 8: pKK indicator

Tracks 9 to 13: Tnp from the pKK-Tnp system (9: control, 10: 0.5 mM

IPTG, 11: 1 mM IPTG, 12: 3 mM IPTG.13: 6 mM IPTG) M: precoloured size marker (P7708S, New England BioLabs)

Figure 9: Graph representing the frequency of transposition and enrichment in transposase, as a function of the culture conditions in the pBadR expression system.

Figure 10: Graph representing the frequency of transposition and enrichment in transposase, as a function of the culture conditions in the pKK expression system.

Figure 11: Graphical representation according to Scatchard of the ITR-binding saturation experiments with MBP-Tnp produced in insect cells. Figure 12: Graphic representation according to Scatchard of ITR-binding saturation experiments with eukaryotic Tnp produced in vitro.

Figure 13: Autoradiogram of an SDS-Page 10% acrylamide gel of the various in vitro syntheses in the presence of ATP-γ ³² P.

1: without DNA 2: with the vector pET-26b +

3: with the vector pET-Tnp (300ng)

Figure 14: Graphical representation according to Scatchard of ITR-binding saturation experiments with dephosphorylated MBP-Tnp.

Figure 15: Graphic representation according to Scatchard of the ITR-binding saturation experiments with Tnp produced in vitro and dephosphorylated. Figure 16: Autoradiograms and graphs illustrating the results of delayed gel analysis (A and C) and transposition into bacteria (B and D) of the transposases by Zhang et al. (2001) (A and B) and mutated transposases at position T88 (C and D). WT: wild transposase

The experimental part below, supported by examples and figures, illustrates, without limitation, the invention.

1. Materials and Methods

1.1 Description of the vectors used:

The vector pBadR-Tnp (5.6 kb) allows the expression of the transposase under the control of a weak, modular promoter, inducible to arabinose (Para) and repressed in the presence of glucose via the protein AraC (coded by this vector). PBadR-Tnp carries an ampicillin resistance gene. Its construction is detailed in paragraph 1.2.1. below.

The vector pKK-Tnp (5.6 kb) allows a strong expression of the transposase via a Plac promoter inducible to IPTG. The promoter is not modular and has a natural expression leak. PKK-Tnp also carries the ampicillin resistance gene. The construction of the vector is presented in paragraph 1.2.2. infra.

PBC 3K3 is a mos-pseudo-mariner donor plasmid (Augé-Gouillou et al, 2001). It contains the kanamycin resistance gene "OFF" (ie without promoter) bordered by two ITR 3 '. Thus, only the bacteria which have carried out the transposition of the pseudotransposon downstream of a promoter (“tagging” promoter) will be selected on LBkanamycin boxes. The vector carries a chloramphenicol resistance gene. PBS (3 ') carries the 3' ITR clone at the Smal site of pBS (Stratagene, La Jolla, California, United States). PBS is identical to pBC, except that it carries an ampicillin resistance gene instead of chloramphenicol.

The vector pET-Tnp (6.4 kb) (Augé-Gouillou et al, 2001) codes for the transposase under the control of the T7 promoter and has a gene for resistance to kanamycin. This vector was used as a source of DNA for in vitro transcription / translation into a reticulocyte lysate to produce a eukaryotic transposase. BL21 bacteria, encoding T7 polymerase under IPTG-inducible Plac control, were transformed with pET-Tnp to produce prokaryotic transposase in the phosphorylation test described below.

The vector pGEM®-T-Easy (Promega, Charbonnières, France) has the primers for sequencing Pu and pRev (Ausubel et al., 1994) and the gene for resistance to ampicillin. It is designed to clone PCR products into the LacZ gene, which allows white / blue screening of the bacterial colonies obtained on an LBampicillin dish, in the presence of IPTG and X-Gal. It was used to give the pGEM-T (Tnp) (Augé-Gouillou et al, 2001) and the pGEM-T (L27) whose construction is detailed in paragraph 1.2.3. below.

1.2 Construction of vectors:

1.2.1 Preparation of the DNA of the vectors: For the various constructions, all the DNA elutions from an agarose gel were carried out with the Geneclean II kit (BIO 101, UK). All mini-preparations of plasmids from bacterial culture were carried out with the Wizard Plus Minipreps kit (Promega). 1.2.2 pBadR-Tnp:

PBadR-Tnp was obtained from pBad 18 in two stages: o Cloning of RBS (Ribosome Binding Site) Cloning of RBS in pBad 18 reconstitutes the following sequence: 5 'GAAGGAGTAcccgggGATC 3' (SEQ ID N ° 3)

This sequence corresponds to a consensus site for initiating translation if the gene encoding the transposase is cloned into a Sma \ site (in lower case). RBS was purchased as two non-phosphorylated complementary single-stranded oligonucleotides. The pairing of the two strands reconstitutes the following sequence: 5 "AATTC GAΓAΓCGAAGGAGTAC 3 '(SEQ ID N ° 4) - 3' G CTATAGCΎTCCI 5 '(SEQ ID N ° 5) The outgoing 5' end corresponds to an EcoRI site, the outgoing 3 'end at a Kpn \ site, allowing the cloning of the RBS in pBad18. This sequence also provides a unique EcoRV site (in italics) which makes it possible to control the cloning.

The two paired strands were phosphorylated with T4 polynucleotide kinase (New England BioLabs, Beverly, MA, United States). The plasmid pBad 18 was digested with Kpn \ and EcoRI, then dephosphorylated. The RBS (15 ng) was then ligated to the plasmid (25 ng) overnight at 16 ° C in the presence of a T4 DNA ligase (Promega), in order to obtain pBadR. Competent E. coli MC1061 bacteria (strain lacking an active transport of arabinose) were transformed with pBadR, on LB dish (5 g NaCl, 5 g yeast extract, 10 g tryptone, 0.3 ml of 10N NaOH, qs 1 L H2O) / ampicillin (100 μg / ml). Twelve ampicillin-resistant colonies were cultured to extract plasmid DNA and analyze the plasmid by EcoRV digestion. In order to verify which plasmids had integrated a single RBS, the plasmid DNAs were sequenced using the “DNA sequencing kit” from Perkin Elmer Biosystems (Courtaboeuf, France) and analyzed on a monolaser automatic sequencer of the Licor type (Science Tec , Ulysses Innovation Park, France). The plasmid containing a single RBS was then used to clone the gene encoding the transposase.

o Cloning of the gene coding for transposase: The fragment coding for transposase (Tnp) was prepared from the vector pGEM-T (Tnp) by SnaBUHind W digestion and eluted on 0.8% agarose gel (TAE 1X: 0.04 M Tris-acetate, 1mM EDTA, pH 8). The plasmid pBadR obtained previously was digested with Sma \ / Hind \\\. After electrophoresis on an agarose gel, the DNA of the plasmid was eluted, then ligated with the fragment coding for the transposase of Mos-1 (called Tnp in this experimental part), at a rate of 25 ng of fragment for 25 ng of vector. The ligation product was used to transform E. coli MC1061 bacteria which were then selected on an LB-ampicillin dish (100 μg / ml). 12 ampicillin resistant clones were cultured for extraction of the plasmid. The DNA mini-preparations were checked by EcoRV / H / r / c / III digestion and then by 0.8% agarose gel electrophoresis (TAE 1X) to ensure that they had integrated the gene encoding the transposase. .

1.2.3 pKK-Tnp:

The fragment coding for the transposase (Tnp) of marinator Mos-1 was prepared from pGEM-T (Tnp) by Nco \ IHind \\\ digestion and eluted on gel. It was ligated with the vector pKK-233-2 (Clontech, Ozyne, Saint-Quentin-en-Yvelines, France), opened by the same enzymes, at a rate of 10 ng of vector for 40 ng of Tnp fragment, to give pKK-Tnp. MC1061 bacteria were transformed with the expression vector pKK-Tnp, spread on an LB-ampicillin dish (100 μg / ml), 50 mM CaCl2 and placed at 42 ° C, to limit the cytotoxic effects of the transposase. 6 clones resistant to ampicillin were cultured under the same conditions to extract the plasmids. Mini DNA preparations have were checked by Nco \ / Hind \\\ digestion on 0.8% agarose gel (TAE 1X) to ensure that they had integrated the gene coding for transposase.

1.2.4 Cloning of the L27 probe: The sequence of the gene coding for the ribosomal protein L27 of E. coli was obtained from the Infobiogen website (www.infobiogen.fr). The primers SEQ ID No. 6 and SEQ ID No. 7, for the amplification of the gene by polymerase chain reaction or PCR (for “Polymerase Chain Reaction”), were drawn with the OLIGOV2 software, then ordered in the form of 'single stranded oligonucleotides.

SEQ ID N ° 6: 5'-ATGGCACATAAAAAGGCTG-3 'SEQ ID N ° 7: 5'-TTATTCAGCTTCGATGCT-3' The L27 gene was amplified by PCR technique from genomic DNA from Eco //. The amplification products were eluted after electrophoresis on 0.8% agarose gel, then ligated into the vector pGEM®-T Easy before being cloned. E.coli competent bacteria (DH5α) were transformed with the ligation mixtures, then spread on LB-ampicillin dishes (100 μg / ml) containing X-Gal (2%) and IPTG (1 mM). 2 white clones resistant to ampicillin were cultured to extract the plasmid DNA. After monitoring the cloning by EcoRI digestion and 0.8% agarose gel electrophoresis (TAE 1X), the plasmid was sequenced using the “DNA sequencing kit” (Perkin Elmer Biosystems) and analyzed on an automatic monolaser sequencer of the Licor type in order to to verify the conservation of the sequence before marking.

1.2.5 Marking of the L27 and Tnp probes:

The L27 probe was labeled by radioactive PCR from the pGEM-T matrix (L27) with the primers pU and pRev (which surround the cloning site [Ausubel et al., 1994]) in the presence of ATP-γ ³² P. The labeled probe is separated from the free dNTPs by size exclusion chromatography on a Sephadex G50 column (TE 1X: 10mM tris pH 8, 1mM EDTA). The column was washed in a fraction of 200 μl and the probe identified with the MIP 10 counter (Eurisys Measurements, Saint-Quentin-en-Yvelines, France).

The Tnp probe was marked by the technique of "Random Priming", ie the synthesis of DNA after pairing of random hexamers. The elongation was carried out with the Klenow fragment of DNA polymerase in the presence of ATP-γ ³² P. The probe was eluted by size exclusion chromatography on a Sephadex G50 column (TE 1X).

1.3 Induction of bacteria containing the vector encoding the transposase

With pBadR-Tnp: With pKK-Tnp:

1% Glucose • Control (without IPTG)

Control (0% glc, 0% ara) • 0.5 mM IPTG

0.001% arabinose • 1 mM IPTG

0.01% arabinose • 3 mM IPTG

0.1% arabinose • 6 mM IPTG 1% arabinose

Induction protocol:

From a saturated culture of E. coli MC1061 bacteria containing the vector of interest (pBadR-Tnp or pKK-Tnp), 50 ml of LB were seeded at 1/100 and cultured for 3 h at 31 ° C. with stirring (250 rpm) in the presence of 1% glucose (Para inhibitor) in the case of pBadR-Tnp. The culture was then centrifuged for 15 min at 3000 rpm and the pellet was taken up in 2.5 ml of LB, in order to wash it and remove the glucose. Bacteria containing pKK-Tnp have the same treatment. The induction tubes (2.5 ml LB containing arabinose or IPTG according to the above conditions) were seeded volume to volume, and cultured for 3 h at 31 ° C (final volume of 5 ml ). After three hours, the proteins and mRNAs were extracted. The same protocol was followed for the bacteria containing the plasmids pBadR and pKK, in order to produce RNA and proteins devoid of Tnp (negative controls).

1.4 Determination of mRNAs

1.4.1 Extraction of bacterial RNA:

The extraction of the mRNAs was carried out at 4 ° C., in the absence of RNase in order to avoid their degradation. Following induction, the bacterial cultures were centrifuged for 10 min, at 12,000 g, at 4 ° C. The supernatant was removed, the residue taken up in 5 ml of “protoplasting buffer” (15 mM Tris pH 8, 15% sucrose, 8 mM EDTA). 40 μl of lysosyme (50 mg / ml) were added and the mixture was incubated for 15 min in ice. After centrifugation for 5 min at 6000 g, at 4 ° C., then removal of the supernatant, the pellet was taken up in 250 μl of lysis buffer (1.5% SDS, 1 mM sodium citrate, 10 mM NaCl, 10 mM Tris pH 8).

After transferring the lysate into 1.5 ml tubes, 7.5 μl of DEPC were added; the mixtures were then incubated for 5 min at 37 ° C. The tubes were cooled in ice and mixed by inversion with 1/2 volume of a saturated NaCl solution. After an incubation in ice for 10 min, the tubes were centrifuged for 10 min at 12,000 g. The supernatants were transferred to 1.5 ml tubes, and the nucleic acids were precipitated with 3 volumes of cold 100% ethanol (EtOH). The tubes were placed for 30 min at -80 ° C or at -20 ° C for 16h. The tubes were then centrifuged for 15 min at 12000 g, at 4 ° C. The supernatants were eliminated, the pellets washed with 500 μl of 70% EtOH, then dried. The pellets (taken up in 30 μl H2O DEPC) were subjected to the action of Dnasel for 1 h 30 at 37 ° C. After phenol-chloroform extraction and precipitation, the RNA pellets were washed and dried, then taken up in 50 μl of H2θ DEPC. The quality of the extraction was checked with 5 μl of sample on 0.8% agarose gel (TAE 1X). The extracted RNAs were stored at -80 ° C.

14.2 Analysis of RNAs:

The amounts of total RNA were assayed using a fluorescence spectrophotometer.

Each extract was diluted to a hundredth in 500 μl of water. The absorbance was measured at 260 nm (wavelength of maximum absorption of nucleic acids) and at 280 nm (wavelength of absorption of amino acids such as tyrosine). If the 260 nM to 280 nM absorption ratio was between 1.8 and 2.1, the total amount of extracted mRNA could be quantified, at the rate of 40 μg for an absorbance measurement at 260 nm in 1 ml. To quantify the transposase mRNAs, a constant quantity of total RNAs (which was calibrated using an RNA control, the RNA coding for the ribosomal protein L27, the quantity of which did not vary during the experiments) was deposited on a hybridization membrane. After thermal fixation (15 min at 80 ° C), the membrane was pre-hybridized with Church buffer (7% SDS, 1 mM EDTA, 0.5 M NaPO4 pH 7.2) in order to limit non-specific hybridizations by saturation of the membrane, for 1 hour at 65 ° C.

Hybridization with the L27 control probe The pre-hybridization buffer was replaced by new “Church” buffer to which 6000 shots per minute (cpm) were added (to the MIP counter 10) of the probe labeled L27. The membrane was hybridized overnight at 65 ° C. The membrane was then washed for 1 h with a 3X SSC buffer (20X SSC: 3M NaCl, 0.3 M sodium citrate) 10.1% SDS and then with a 1X SSC / 0.1% SDS buffer. The presence of L27 mRNAs was detected and quantified at the Direct Imager (Packard, United States) in cpm. A constant quantity of L27 mRNA for each deposit should make it possible to confirm the constant quantity of total RNA deposited. Otherwise, it should make it possible to correct the quantities of Tnp mRNA.

The membrane was then dehybridized by three rinses with a 1% SDS solution brought to a boil. The membrane was then again put in pre-hybridization for 1 h at 65 ° C. before a new hybridization.

Hybridization with the Tnp probe The pre-hybridization buffer has been replaced by buffer "

New Church ”to which 6000 cpm of the Tnp probe was added. The membrane was hybridized overnight at 65 ° C. The membrane was then rinsed for one hour with a 3X SSC / 0.1% SDS buffer and then with a 1X SSC / 0.1% SDS buffer. The presence of mRNA-Tnp was detected with the Direct Imager: the quantity of mRNA-Tnp present in the deposits was expressed in cpm.

1.5. Protein determination

1.5.1 Protein extraction:

Bacteria have undergone the same induction protocol as that used for mRNA extractions. The cultures were centrifuged for 10 min at 12,000 g at 4 ° C. The bacterial pellets were taken up in 500 μL of a 20 mM Tris pH 9 buffer, 100 m M NaCl, 1 mM DTT, and stored at -20 ° C. overnight. After sonication (1 "draws" of 30s at 25 W) and centrifugation at 10,000g, 4 ° C, for 15 min, the protein supernatants were collected and then quantified by a Bradford assay relative to a concentration of bovine serum albumin (BSA). After dosing, the protein extracts were stored at -20 ° C.

1.5.2. Protein analysis in SDS-PAGE:

Sample preparation and electrophoresis:

A constant amount (12 μg) of protein was analyzed. A volume of loading buffer was added to each extract, the whole was denatured at 100 ° C. for 5 min. The samples were deposited in a gel

SDS-PAGE 10% acrylamide [375 mM Tris HCI pH 8.8, 0.1% SDS, '10% acrylamide (30: 0.8)] 10%. After an electrophoresis over 16 h at 50 V, the gel was fixed in a 2% H3PO4-50% CHtaOH solution with gentle stirring for 2 to 12 h.

Coloration of the colloidal Coomassie blue gel:

The gel was washed in water 3 times 30 min. This step was repeated with a 2% H3PO4 solution.

The gel was then placed in a 17% CH3OH, 2% H3PO4 and 15% (NH4) 2SO4 solution for one hour. The gel was then colored overnight in an identical fresh solution, to which was added 0.2% of colloidal Coomassie blue. The colored gel was scanned with a scanner and then dried.

1.5.3. Late gel analysis of the activity of the extracted transposases:

This method makes it possible to test the binding activity to ITR 3 ′ (ITR optimized for the binding of Tnp) of the proteins produced. The 3 'ITR was isolated from the plasmid pBS (3') by EcoR \ / Xba \ digestion, purified on a 2% NuSieve agarose gel (FMC products, United States), eluted and quantified. 100 ng of 3 'ITR were labeled with the Klenow fragment of DNA polymerase in the presence of dATP. After phenol extraction chloroform and precipitation, the pellet was taken up in 40 μl of water to obtain the ITR at a concentration of 50 nM.

5 μg of crude extracts were incubated, with the labeled 3 'ITR, under the following conditions: 1 nM of probe, 1 μg of sonicated salmon sperm DNA, 5 mM MgC, 5% glycerol, 20 mM Tris pH9, for 15 min in ice. Each sample was deposited on a 9% hydrolink gel (Long Ranger, TEBU, Le Perray en Yvelines, France) (0.25X TBE: 22.25 mM Tris, 22.25 mM boric acid, 2.5 mM EDTA disodium dihydrate). After electrophoresis and drying of the gel, the delay was quantified with the Direct Imager (Packard). Autoradiography of the gel was then performed.

1.6. Transposition test

Induction conditions: With pBadR-Tnp: With pKK-Tnp:

1% Glucose Control Control 0.5 mM IPTG

0.001% arabinose 1 mM IPTG 0.01% arabinose 3 mM IPTG 0.1% arabinose 6 mM IPTG 1% arabinose

The principle of the transposition test is illustrated in Figure 3.

Competent bacteria MC1061 were co-transformed with the plasmid pBC 3K3 (carrying a pseudo-mar / ner reporter of the transposition and of the gene for resistance to chloramphenicol) and with the inducible vector coding the transposase pBadR-Tnp or pKK- Tnp (carrying the ampicillin resistance gene) (Figure 3A). The bacteria were selected on ampicillin and chloramphenicol to verify the presence of two plasmids. Co-transformation was also carried out with the control plasmids pBadR and pKK. The E. coli MC1061 bacteria, containing the reporter vector pBC3K3 and that encoding the transposase, were cultured, at 42 ° C., in 2.5 ml of LB-ampicillin (100 μg / ml) chloramphenicol (20 μg / ml ) / 50 mM CaCI ₂ overnight (conditions limiting the activity of transposase). The culture was titrated on an LB dish (10 μl of a dilution allowing correct counting), and on an LB-kanamycin dish (100 μg / ml) (50 μl of the saturated culture). 2.5 ml of LB (containing arabinose or IPTG as indicated above) were seeded at 1/100 and cultured for 3 h, at 31 ° C (optimum operating temperature of the transposase) and under shaking at 250 rpm (Figure 3B). After 3 hours of induction, the culture was spread on an LB dish (bacteria titration) and on an LB-kanamycin dish (listing of transposition events) (FIG. 3C). The boxes were placed at 37 ° C overnight. The next day, the colonies were counted on the LB and LB-kanamycin dishes, then the transposition frequency, equal to the total number of bacteria resistant to kanamycin, was calculated on the total number of bacteria.

1.7. Equilibrium binding saturation experiments: Scatchard test The principle of this test is to determine the affinity (Kd) of the transposase produced in the eukaryotic system for ITR 3 '(ligand), thanks to saturation of the equilibrium bond behind gel. The results are represented according to the Scatchard method. For a constant quantity of protein extracts, the quantity of labeled 3 'ITR was varied within a concentration range framing the Kd. After an incubation of 15 min in ice (carried out under conditions identical to those of the gel delay experiments), the ITR-protein complexes were deposited on a native 9% acrylamide gel (Long Ranger, TEBU) (TBE 0.25X ). After electrophoresis and drying, the gel was analyzed with Direct Imager (Packard). The amount of ITR bound to the protein and the total amount of ITR were measured in counts per min (cpm). To determine the dissociation coefficient (Kd):

The total amount of ITR bound to transposase (B) was subtracted from the total amount of ITR present (T) to give the amount of free ITR (F). The B / F ratio expressed as a function of the nM of bound ITR (nM B) gave a straight line with a slope of -1 / Kd.

This test was carried out with a fusion protein, MBP-Tnp (given by Dr S. Tourne) produced in insect cells using a baculovirus vector and with the transposase produced in a eukaryotic in vitro transcription / translation system. (TNT® T7 Quick Coupled Transcription / Translation System, Promega) with the vector pET-Tnp. This test was also carried out with Tnp and MBP-Tnp (from eukaryotic cells) having undergone a dephosphorylation of 30 min at 30 ° C with calf intestine phosphatase (CIP).

1.8. Phosphorylation tests

1.8.1. Prokaryotic test:

From a preculture of BL21 bacteria containing the pET-Tnp vector, 25 ml of LB were seeded at 1/100 in the presence of inorganic phosphorus (PiP ³³ ) for 3 h at 37 ° C. Transposase synthesis was then induced in the presence of 1mM IPTG for three hours at 37 ° C. Proteins extracted from this culture were analyzed on a 10% SDS-PAGE gel. After 16 hours electrophoresis at 50V, the gel was stained with colloidal Coomassie blue. After drying, the gel was placed in cassette for autoradiography, in order to visualize the phosphorylated proteins.

1.8.2. Eukaryotic test: The plasmid pET-Tnp was used in a eukaryotic (rabbit) in vitro transcription / translation system (TNT® T7 Quick Coupled Transcription / Translation System, Promega) to allow the synthesis of transposase, identical to that produced previously in prokaryotes. This system allows protein phosphorylation. The possible phosphorylation of the transposase had to be visualized thanks to the addition of ATP-γ ^ P during the synthesis.

0.5 to 1 μg of pET-Tnp were incubated for 90 min at 30 ° C with 40 μl of TNT mix in the presence of ATP-γ ³² P. 5 μl of the mixture was denatured with the same volume of loading buffer , then deposited in a 10% acrylamide SDS-PAGE gel. After electrophoresis, the gel was stained with colloidal Coomassie blue and then dried. The gel was autoradiographed to reveal the presence of the phosphorylated protein.

2. Results

The Mos-1 transposon mariner has a low transposition activity in its natural host (D. mauritiana), compared to the P element (Rubin et al, 1982). In order to be able to analyze all the stages of the transposition of marinator Mos-1 into a simpler system than eukaryotic cells, a study model in bacteria has been developed. This model revealed that the transposition of a Mos-1 element carrying two 3 'type ITRs was 10,000 times more efficient than that of an element in a “natural” configuration, that is to say carrying an ITR 5 'and an ITR 3'. This difference is observed in prokaryotes but not in eukaryotes, as shown by the work carried out in Drosophila by Dr P. Atkinson (University California Riverside) and in HeLa cells by Dr S. Tourne (University of Tours) (personal communications). The results obtained in this context on eukaryotic cells revealed a very low transposition activity, with the two marinating elements (3 '/ 3' and 5 '/ 3'). In order to understand why the Mos-1 marinator transposase behaves differently in the prokaryotic system, the steps limiting the activity of transposase in eukaryotes have been identified. Then, the differences between the prokaryotic and eukaryotic transposase that cause the reduction in the efficiency of the transposition when produced in the eukaryotic system were determined. For this, two early stages of transposition were tested: protein production and binding to ITR. Finally, a possible mode of regulation of the Mos-1 transposase, phosphorylation, was tested.

2.1. Inhibition of transposition by overproduction of transposase

Lohe and Hartl (1996) have shown that the transposition frequency decreases in eukaryotes when the marin-transposase Mos-1 is overexpressed. This phenomenon has been called OPI for "Over Production Inhibition". In what follows, the expression Inhibition by Overproduction (IPS) will be used.

In a first series of experiments, we wanted to know if the transposase produced in the bacterial system was subjected to IPS. For this, two expression systems were used to analyze the variations in the transposition frequency of the Mos-1 marinator as a function of the amount of transposase present in the bacteria. The vector pBadR-Tnp is a weak expression system where the transposase is under the control of a promoter inducible to arabinose (Para) and modular, that is to say that the quantity of protein produced is a function of the amount of inductor. PKK-Tnp is a strong expression system where the transposase is under the control of a promoter inducible to IPTG (Plac) and non-modular.

2.1.1. Correlation level of induction / quantity of mRNA / quantity of transposase: In order to validate the expression vectors, it was ensured that the quantities of Tnp mRNA and Tnp proteins were well correlated with induction conditions. PBadR-Tnp was cultured in the absence of an inducer (control level), in the presence of glucose (Para repressor) and with a range of 0.001% to 1% of arabinose (Para inducer). PKK-Tnp was cultured in the absence of an inducer (control level) and in the presence of a range of 0.5 mM to 6 mM IPTG (Plac inducer).

1) Characterization of the mRNAs After induction and extraction, the mRNAs were hybridized with a control probe for the ribosomal protein L27 in order to calibrate the quantities of RNA extracted under the different culture conditions, then with a transposase probe in order to quantify the level mRNA-Tnp (Figures 4 and 6) at cpm. The quantities of Tnp-mRNA detected (converted as a percentage relative to the maximum quantity detected) are represented (Y axis) as a function of the different culture conditions (X axis) in FIGS. 5 and 7.

Analysis of the pBadR-Tnp expression system:

In the weak expression system (pBadR), transposase mRNAs were absent in bacteria containing the control vector pBadR. On the other hand, they were present in bacteria containing the expression vector pBadR-Tnp: the lowest amounts of mRNA-Tnp were detected in the absence of arabinosis or in the presence of glucose, and greatest amounts were observed at all induction conditions with a maximum amount of Tnp-mRNA for 0.1% arabinose (Figures 4 and 5). These results indicated that the Para promoter was not completely repressed by glucose and that it exhibited expression leakage in the absence of glucose / arabinose under the culture conditions described here. The amount of mRNA encoding the transposase followed the inductor range, except for the 1% arabinose point. The correlation between the quantity of Tnp-mRNA and the induction conditions in the pBadR system has shown that this expression system was consistent with the known functioning of the arabinose-inducible promoter (Para). Only the 1% arabinose induction point resulted in a decrease in the amount of transposase mRNA. This difference could be explained by the high concentration of arabinose used for induction, which had to saturate or even inhibit the induction system. Another phenomenon could also explain this result: a repression of the system by reducing the number of copies of the plasmid pBadR-Tnp in the presence of high concentration of arabinose. The presence of Tnp mRNA in glucose condition (Para repressor) showed that the promoter was not fully lockable, which had already been shown by Guzman et al. (1995). In addition, the bacteria were cultivated in a rich medium (LB) whose exact composition is not known (yeast extracts and tryptone), the medium could interact with the normal functioning of the promoter, in particular with respect to expression of the AraC gene coding for the Para repressor.

Analysis of the pKK-Tnp expression system:

In the strong expression system (pKK), the mRNAs encoding the transposase were absent in the bacteria containing the pKK control vector and present for the bacteria containing the pKK-Tnp vector: weakly in the absence of an inducer, strongly in the presence of IPTG. The maximum amount was detected from 0.5mM IPTG (Figure 7). In a strong expression system (pKK-Tnp), the amount of Tnp-mRNA synthesized there again reflected the known functioning of the Plac promoter inducible to IPTG. This promoter, in the absence of an inducer, was not completely repressed, which resulted in a synthesis of mRNA-Tnp (Figures 6 and 7). On the other hand, in the presence of IPTG, the promoter was active, which resulted in a strong expression: whatever the concentration of IPTG, the quantity of mRNA-Tnp remained constant. 2) Protein analysis:

The proteins were obtained under the same conditions as the RNAs. They were analyzed on SDS-PAGE (Figure 8A). The digital image of the gel made it possible to calculate the percentage of transposase in the different extracts using the Molecular Analyst software (Biorad, Ivry sur Seine, France). At the same time, the binding activity to the ITR 3 'of the transposases obtained in the different extracts was tested, by gel retardation experiments (FIG. SB). The graphs (Figures 9 and 10) represent the percentage of transposase in the extracts (relative quantities) (y axis) as a function of the different induction conditions (x axis). The Tnp mRNA levels were replaced there in order to correlate mRNA and protein.

Analysis of the pBadR-Tnp expression system: The proteins extracted from the bacteria containing the pBadR control vector did not contain transposase as shown in the protein gel (FIG. 8A, lane 1) and the late gel analysis (FIG. 8B , track 1). The extracts obtained from bacteria containing the vector pBadR- Tnp (FIG. 8A, tracks 2 to 7) contained very small amounts of transposase in the absence of inducer (2.42% of the crude extract, track 3) or in presence of glucose (1.04% of the raw extract, lane 2). The quantities obtained in the presence of inductor were slightly higher (2.62% to 3.1% of the crude extract, lanes 4 to 7). Figure 5 shows that the amount of transposase was well correlated with the amount of Tnp mRNA, except for the strongest point of induction (1% arabinose) where the amount of protein increased while the amount of mRNA decreased. This could be explained by the accumulation, at the start of induction, of the transposases synthesized before the quantity of mRNA is regulated. The late gel analysis (Figure 8B) shows that there was no detectable delay, even for high concentrations of crude extracts and for large quantities of inducer. The amounts of transposases produced with the expression vector pBadR-Tnp were therefore very low, which was in agreement with the activity of the arabinose-inducible promoter, and could therefore explain the absence of signal during delay experiments. on gel. Only the transposition test (biological test) could confirm or not the activity of the transposases produced with this system.

Analysis of the pKK-Tnp expression system:

The proteins extracted from the bacteria containing the pKK control vector did not contain transposase, as indicated by the protein gel (lane 8 in FIG. 8A) and the late gel analysis (lane 8 in FIG. 8B). The extracts obtained from bacteria containing the vector pKKTnp (FIG. 8A, lane 9 to 13) contained small amounts of transposase in the absence of IPTG (2.37% of the crude extract, lane 9) and large amounts of transposase in the presence of IPTG (8.48% to 10.25% of the crude extract, lane 10 to 13). FIG. 7 shows the level of synthesized Tnp-mRNA and of transposase produced as a function of the induction conditions: the quantity of Tnp produced exhibited the same evolution as the quantity of mRNA, the minimum quantity was obtained in the absence of inducer , the maximum regardless of the quantity of inductor. ITR binding activity was strongly detected late on gel when the amount of transposase was high (lanes 10 to 13 in FIG. 8B). The Mos-1 mariner element transposase produced in the pKK-Tnp system always seemed to be active, whatever the induction conditions (FIG. 8B). Large amounts of transposase therefore did not appear to affect the ability to bind to ITR 3 '.

These first results made it possible to conclude that the quantities of RNA-Tnp produced / the quantity of transposases produced in the two expression systems were correlated. 2.1.2. Correlation of transposase quantities / transposition frequencies: The transposition test was carried out under the same conditions as those which made it possible to obtain proteins and mRNAs. The only difference was the additional presence in bacterial strains of a transposition reporter vector (pBC 3K3). This transposition test made it possible to know whether an increase in the amount of transposase resulted in a decrease in the frequency of transposition, as would be expected if the prokaryotic transposase was subjected to IPS.

Analysis of the pBadR-Tnp expression system:

Analysis of transposase production showed that the protein was present in our cultures, including in the absence of an inducer (arabinose) or in the presence of a repressor (glucose). Transposition began even in the absence of induction (Figure 9, glucose conditions and control). This confirmed that, even in small quantities, the transposase had a high activity. Indeed, for a minimum quantity of transposase (1.04% of the crude extract in glucose condition), the transposition frequency was 26.10 ^"5. For a transposase enrichment of 2.62% of the crude extract ( induction point 0.001% arabinose), the transposition frequency, 68.10 ^"5 , was maximum, a slight increase by a factor of 2.5. For the induction points of 0.01% to 1% arabinose, the transposition frequency remained at a plateau despite a low enrichment in transposase compared to the induction point 0.001% arabinose.

The transposition experiments in the pBadR-Tnp system therefore indicated that the transposase, even produced in small quantities, was still active. In addition, the maximum transposition frequency was reached at the first induction point (0.001% arabinose), then reached a plateau for the following induction points. Inhibition By Overproduction (IPS) was therefore not observed in the pBadR-Tnp system, which seemed normal given the little transposase produced.

Analysis of the pKK-Tnp expression system: Analysis of the protein production showed that the transposase was produced whatever the culture conditions (FIG. 7, lanes 9 to 13). FIG. 10 represents the transposition frequency as a function of the induction conditions. The transposase enrichments have been added in order to correlate them with the transposition frequencies. For a minimum amount of transposase (in the absence of induction), the transposition frequency was 8.10 ^"5. The maximum transposition frequency was 59.10 ^{" 5} for a transposase enrichment of 8.48% (corresponding to the first point d 'induction 0.5mM IPTG), then reached a plateau for amounts of transposase varying negligibly (8 to 10% of crude extracts for the following induction points). These results therefore indicated a saturation of the production system from the first induction point, which corresponded to the functioning of the promoter which is highly inducible at IPTG. In the pKK-Tnp system, the phenomenon of IPS was not observed, despite the presence of high levels of transposase in the crude extracts.

2.1.3. Conclusion on IPS:

The amount of transposase produced in the pBadR-Tnp expression system was small but sufficient to have an efficient transposition, even in the absence of an inducer. In the pKKTnp system, the production of transposase represented up to 10% of bacterial protein and made it possible to detect its activity by a biochemical test (delayed gel). However, this higher production did not translate into a higher transposition frequency compared to the pBadR system. From the first induction point (0.5 mM IPTG), the transposition frequency was maximum (59.10 ^"5 ) and similar to that obtained with pBadR-Tnp (68.10 ^"5 ) for the induction point 0.001% arabinose. This frequency stagnated for the other induction points. Unlike the eukaryotic system, all of these results seemed to indicate that in the prokaryotic system , no inhibition by overproduction was detected, which should result in a drop in the transposition frequency.

2.2. Transposase affinity for ADW

There was therefore a first difference in behavior between the transposase of the mariner element Mos-1 produced in the eukaryotic and prokaryotic system: IPS seemed to intervene only in the eukaryotic system. In order to find other possible levels of regulation of transposition, the quality of binding between the 3 ′ end of the mos-1 transposon (ITR 3 ′) and the transposase was tested. In fact, this step is the first step in the transposition process. To determine the affinity of the transposase for ITR (measurement of Kd), experiments on saturation of the equilibrium binding were carried out and analyzed late on gel (FIGS. 11 and 12, boxes). These experiments were carried out with a constant amount of transposase in the presence of a concentration range of ligand (therefore a concentration range of ITR 3 '). The results obtained were represented according to Scatchard. In this type of representation, the ratio of the amount of ligand (ITR) bound to the amount of free ligand (B / F) is expressed as a function of the concentration of bound ligand (nM B) and is visualized by a line of equation y = ax + b, where a = -1 / Kd.

These tests, already carried out on the transposase produced in the prokaryotic system, made it possible to calculate a dissociation coefficient (Kd1) of 0.6 nM for binding to the ITR 3 '(publication submitted). A second Kd (Kda = 10 nM) measured the binding of the ITR as an integration target. The same type of test was carried out with two Mos-1 mariner transposases produced in a eukaryotic system: the fusion protein MBP-Tnp given by Dr S. Tourne (Tnp coupled with a protein allowing its purification but having no impact on its activity) produced in insect cells thanks to a baculovirus system and extracted from the cell nucleus, and the transposase produced in reticulocyte lysate (rabbit) using the TNT ® T7 Quick Coupled Transcription / Translation System kit (Promega).

The first experiment was carried out with MBP-Tnp under the same conditions as those which were carried out with transposase of bacterial origin, that is to say for a range of ITR 3 'concentration of 0.05 nM to 10 nM. The results showed that these conditions were not suitable for determining a Kd (the ligand concentration range had to "frame the Kd"), and therefore that the eukaryotic protein behaved differently from the prokaryotic transposase.

Consequently, the range of ITR 3 'has been increased by placing it between 2nM and 50nM. FIG. 11 represents the ratio of bound probe to free probe (B / F) as a function of the concentration of bound probes (nM B), which resulted in a single straight line whose slope was -0.0256. The Kd being the inverse of the slope, its value was 39 nM. Another Scatchard was performed under the same conditions with the transposase produced from pET-Tnp with the TNT® system allowing transcription / translation in vitro in rabbit reticulocyte lysates. The result obtained (FIG. 12) showed a profile identical to that obtained with the transposase produced in insect cells and presented a unique Kd of 74 nM. This result indicated that the transposase produced in vitro in reticulocyte lysate had a binding affinity to ITR 3 'similar to a transposase produced in vivo in insect cells. This in vitro synthesis kit therefore made it possible to produce a transposase having the same behavior as that produced in insect cells. With the two eukaryotic transposases, a real difference in affinity for ITR 3 ', of about a factor of 100, was observed: the transposase produced in bacteria had a strong affinity for ITR 3 ', while the transposase produced in insect cells or in the eukaryotic system in vitro had a weak affinity.

2.3. Impact of phosphorylation on transposase activity

Differences in the behavior of the transposase were highlighted depending on whether it was produced in prokaryotic cells or in eukaryotic cells. In order to determine the origin of these differences, post-translational modifications likely to have an impact on the behavior of transposase were sought.

Starting from an assumption that the transposase could potentially undergo, among other post-translational modifications, phosphorylations, it was first investigated whether the transposase was phosphorylated during its synthesis in eukaryotes and not phosphorylated in bacteria. In fact, bacteria do not phosphorylate proteins of exogenous origin.

2.3.1. Transposase phosphorylation in prokaryotes and / or eukaryotes; Bacteria containing the pET-Tnp vector were cultured in the presence of inorganic phosphorus (PiP ³³ ) so that it is possibly integrated into the proteins synthesized. The analysis in SDS-PAGE gel stained with colloidal Coomassie blue revealed the presence of a band corresponding to the transposase for the bacteria containing the vector pET-Tnp. Once the gel was dried, it was autoradiographed to verify the absence of transposase phosphorylation in prokaryotes. To study phosphorylation in the eukaryotic system, the eukaryotic in vitro transcription / translation kit was used in particular to produce a transposase with activity analogous to that produced in vivo in insect cells. The production of transposase with pET-Tnp, in the reticulocyte lysate, in the presence of ATP- _γ ³² P resulted in the appearance of radiolabelled bands on the protein gel (FIG. 13, lane 3), also present when the experiment was carried out in the absence of DNA (FIG. 13, lane 1) or with pET-26b + (Figure 13, lane 2). A radiolabelled band, present only in the case of production with pET-Tnp (lane 3 in FIG. 13), was located at a PM of around 40kDa, similar to that of transposase.

The results of these two experiments indicated that: the transposase was not phosphorylated when it was produced in bacteria while it was phosphorylated when it was produced in eukaryotic cells.

2.3.2. Impact of phosphorylation on the affinity of transposase for DNA

In order to know whether phosphorylation was involved in the difference in activity between the prokaryotic and eukaryotic transposase, the two proteins produced in the eukaryotic system were dephosphorylated. A Scatchard test was then carried out with a wide range of ligand (0.05 to 25 nM of 3 'ITR), in order to be sure of framing the Kd (s). FIGS. 14 and 15 show a profile similar to that obtained with the transposase produced in the prokaryotic system, that is to say the presence of 2 lines, therefore of 2 different Kd. With MBP-Tnp produced in insect cells and dephosphorylated (Figure 14), the first Kd (Kdi) was 0.66 nM and the second (Kd2) was 24.69 nM. These values were similar to those obtained with the “prokaryotic” transposase (Kdi of 0.6 nM and Kd2 of 10 nM). With the “eukaryotic” transposase produced in vitro (FIG. 15), the Kdi was 1.3 nM and the Kd2 15.5 nM. Again, these values were in the same range as those obtained with the "prokaryotic" transposase. 2.4 Impact of a mutation making the transposase non-phosphorylable at at least one site, on the transpositional activity thereof.

2.4.1. Preliminary results

In accordance with the results described in paragraph 2.2 above, a transposase of bacterial origin is approximately 100 times more effective in recognizing ITRs and initiating transposition than a transposase of eukaryotic origin. Post-translational phosphorylations therefore affect the affinity of the transposase for DNA (specific and non-specific) and limit the conformational activation thereof.

Since the specific binding of each transposase monomer to each of the two ITRs of the transposon is reduced by a factor of approximately 100, the reduction in transposition efficiency caused by phosphorylation, at least at the T88 position, is evaluated as being by a factor approximately equal to the square of 100.

The mutation of the threonine residue at position 88 into a non-phosphorylatable alanine residue, according to a conventional point mutagenesis method (Ausubel et al., 1994), seems to increase the ability of the mutated transposase to catalyze the transposition and transfer of DNA, by a factor of between 100 and 10,000 approximately, compared to that of a native transposase.

2.4.2. Impact of a mutation making the transposase non-phosphorylatable at at least one site, on transpositional activity: importance of a conservative substitution

As illustrated in Figure 16 (A and B), the results of the analyzes carried out on the mutant transposases described in Zhang et al. (2001) show that the S104P transposase is unable to bind to ITRs and to mediate transposition into bacteria. About the transposase S302P, the results show that it is not capable of forming all of the ITR-binding complexes. In addition, she is also unable to mediate the transposition.

The same analyzes were performed on mutated transposases in a reasoned manner at position T88 (Figure 16, C and D).

This position was chosen as far as it has been shown, using antibodies against phosphorylated serines (Qiagen), phosphorylated threonines (Qiagen) and the SQ and TQ dipeptides phosphorylated on serine or threonine (New England Biolabs) , that T88, as well as positions S99 and S104, are phosphorylated in eukaryotes.

The results of the analyzes of the impact of the various mutations at the T88 position show that only the T88V transposase is capable of retaining an ability to mediate the transposition which is not significantly different from that of the native transposase. In addition, they also show that mutations, such as that of the T88E and T88R transposases, greatly reduce the formation of the larger complexes and completely inhibit the ability to mediate transposition. Finally, these experiments reveal that, among the amino acids closest to a threonine (A, C and V), only substitution with a valine is truly conservative.

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Claims

1. A hyperactive mutant transposase of a mobile genetic element (EGM), having at least one mutation by conservative substitution of at least one phosphorylatable residue in at least one phosphorylation site, said conservative substitution making said site non-phosphorylable in vivo, said phosphorylatable residue being chosen from:

- The following residues of the sequence SEQ ID No. 2: T11; T24; S28; T42; T88; S99; S104; T135; S147; T154; S170; T181; S200; T216; T255; and S305; or

the corresponding residues of the sequence of an EGM mariner transposase aligned with said sequence SEQ ID No. 2, said corresponding residues being chosen from:

- a serine; and - a threonine.

2. Transposase according to claim 1, having at least one mutation by conservative substitution of at least one phosphorylatable residue in each phosphorylation site, said conservative substitution making each site non-phosphorylable in vivo.

3. Transposase according to claim 1 or 2, characterized in that said phosphorylatable residue is substituted by a non-phosphorylatable residue.

4. Transposase according to any one of claims 1 to 3, characterized in that it is hyperactive for at least one function from: specific and non-specific binding to DNA, dimerization, transfer of DNA strands , and the endonuclease and nuclear internalisation properties.

5. Transposase according to any one of claims 1 to 4, characterized in that said mobile mariner genetic element belongs to the mauritiana subfamily.

6. Transposase according to any one of claims 1 to 5, characterized in that said mobile mariner genetic element is mos-1.

7. Transposase according to claim 6, characterized in that said phosphorylatable residue is chosen from the following residues (by reference to the sequence SEQ ID No. 2): T11; T24; S28; T42; T88; S99; S104; T135; S147; T154; S170; T181; S200; T216; T255; and S305.

8. Transposase according to claim 7, characterized in that said phosphorylatable residue is T88.

9. Transposase according to claim 7 or 8, characterized in that said phosphorylatable residue is S104.

10. Recombinant nucleotide sequence encoding a transposase according to any one of claims 1 to 9.

11. Recombinant vector characterized in that it comprises at least one recombinant nucleotide sequence according to claim 10.

12. Recombinant vector according to claim 11, characterized in that it allows the expression of said recombinant nucleotide sequence.

13. Recombinant host cell characterized in that it hosts at least one recombinant vector according to claim 11 or 12.

14. A method of producing a transposase according to any one of claims 1 to 9, said method comprising at least: a) cloning of the recombinant nucleotide sequence encoding said transposase in an expression vector; b) transforming a host cell with said recombinant expression vector; and c) expression of said nucleotide sequence by said recombinant host cell.

15. The method of claim 14, further comprising a prior step of obtaining said recombinant nucleotide sequence, by substitution, in the nucleotide sequence encoding the corresponding native transposase, of at least one nucleotide of a triplet encoding a phosphorylatable residue , with another nucleotide, so that the resulting triplet encodes a non-phosphorylatable residue.

16. Method according to claim 14 or 15, characterized in that said host cell is chosen from cells of bacteria, yeasts, fungi, plants, insects and mammals.

17. A method according to any one of claims 14 to 16, further comprising a subsequent step of purification of said transposase.

18. Use of at least one transposase according to any one of claims 1 to 9, for the in vitro transposition of a transposable DNA sequence of interest into a target DNA sequence.

19. Use of at least one transposase according to any one of claims 1 to 9, for the preparation of a medicament intended for allow in vivo transposition of a transposable DNA sequence of interest into the host genome.

20. Use of at least one transposase according to any one of claims 1 to 9, for the in vivo transposition of a transposable DNA sequence of interest in the genome of the host.