WO2004078981A1 - TRANSPOSASES D'ELEMENTS GENETIQUES MOBILES mariner MUTANTES, NON PHOSPHORYLABLES ET HYPERACTIVES - Google Patents
TRANSPOSASES D'ELEMENTS GENETIQUES MOBILES mariner MUTANTES, NON PHOSPHORYLABLES ET HYPERACTIVES Download PDFInfo
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Definitions
- 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.
- 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 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.
- ITR inverted repeat
- 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.
- class I elements or retrotransposons, transpose via the reverse transcription of an RNA intermediate.
- class II elements transpose directly from a site chromosome to another, via a DNA intermediary, according to a “cut and paste” type mechanism.
- TEs 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.
- class II elements include five families: P, PiggyBac, hAT, helitron and Tel-mariner.
- the mobile mariner genetic elements constitute a large group of TE class II, belonging to the Tel-mariner superfamily (Plasterk et al., 1999).
- 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.
- 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:
- MLE are small transposons, easy to handle.
- transposase 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).
- MLE is characterized by a very wide ubiquity in prokaryotes and eukaryotes.
- Dmmarl also called Mos-1
- Mos-1 Drosophila Mauritiana
- Transposition events can be controlled by various factors, such as temperature and pH.
- MLE transposases are phosphorylated in eukaryotes, by means of post-translational modifications (see Experimental Part below).
- the invention aims to suppress, by point mutagenesis, one or more phosphorylation sites of these enzymes.
- the subject of the present invention is a mutant and hyperactive MLE transposase.
- 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.
- such a transposase will be designated as being “mutant, non-phosphorylable and hyperactive”.
- hypoactive 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.
- 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).
- 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.
- first mechanism non-specifically
- second mechanism specifically via ITRs
- these two link modes depend on the conformational state of the N-terminal domain.
- 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).
- 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.
- MLE transposases have two potential bipartite sites allowing nuclear internalization.
- the terms and expressions “activity”, “function”, “property”,
- transposase activity or “transposase function”.
- 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.
- 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.
- 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.
- 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.
- 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.infobio ⁇ en.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).
- the phosphorylation sites targeted by the invention can be conserved through at least two MLE transposases.
- 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".
- corresponding residues are called here "corresponding residues".
- 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.
- 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.
- 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%.
- 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%.
- 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.
- 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)]:
- a mutant, non-phosphorylatable, hyperactive transposase which is the subject of the invention comes from MLE mos-1.
- one or more phosphorylatable residues from the following residues are substituted with non-phosphorylatable residues.
- 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.
- the present invention relates to a recombinant nucleotide sequence encoding a transposase as described above.
- 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).
- the nucleotide and nucleic acid sequences of the invention are DNA.
- the present invention relates to a recombinant vector comprising at least one recombinant nucleotide sequence encoding a transposase according to the invention.
- such a recombinant vector allows the expression of said recombinant nucleotide sequence.
- the subject of the present invention is a recombinant host cell hosting at least one recombinant vector as described above.
- 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).
- a cell suitable for implementing the invention can come from a bacterium (e.g., Escherichia coli).
- the present invention relates to a process for the production of a mutant, non-phosphorylatable, hyperactive transposase, as described above.
- 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).
- 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.
- 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).
- a subsequent step of purification of said transposase is carried out.
- 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".
- pure enzyme or pure transposase
- 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).
- the host cell is chosen from prokaryotic cells, for example from bacteria, and eukaryotic cells, for example from yeasts, fungi, plants, insects and mammals.
- the present invention relates to uses of at least one mutant, non-phosphorylatable, hyperactive transposase, as described above, in the field of biotechnology.
- such a transposase is used for the in vitro transposition of a transposable DNA sequence of interest into a target DNA sequence.
- 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.
- 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.
- 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.
- 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:
- 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 ⁇ sp 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.
- 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:
- 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)
- IPTG 11: 1 mM IPTG, 12: 3 mM IPTG.13: 6 mM IPTG
- 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- ⁇ 32 P.
- 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
- 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, Charbonippos, 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.
- 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 GA ⁇ A ⁇ CGAAGGAGTAC 3 '(SEQ ID N ° 4) - 3' G CTATAGC ⁇ TCCI 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).
- 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 ⁇ g / ml). Twelve ampicillin-resistant colonies were cultured to extract plasmid DNA and analyze the plasmid by EcoRV digestion.
- 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.
- Tnp 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).
- TEE 1X 0.04 M Tris-acetate, 1mM EDTA, pH 8.
- the plasmid pBadR obtained previously was digested with Sma ⁇ / Hind ⁇ .
- 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 ⁇ g / 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. .
- 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 ⁇ g / 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.
- 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.
- 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- ⁇ 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 ⁇ l 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- ⁇ 32 P.
- the probe was eluted by size exclusion chromatography on a Sephadex G50 column (TE 1X).
- 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).
- 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 ⁇ l of lysosyme (50 mg / ml) were added and the mixture was incubated for 15 min in ice.
- “protoplasting buffer” 15 mM Tris pH 8, 15% sucrose, 8 mM EDTA.
- the pellet was taken up in 250 ⁇ l of lysis buffer (1.5% SDS, 1 mM sodium citrate, 10 mM NaCl, 10 mM Tris pH 8).
- RNA pellets were washed with 500 ⁇ l of 70% EtOH, then dried.
- the pellets (taken up in 30 ⁇ l H2O DEPC) were subjected to the action of Dnasel for 1 h 30 at 37 ° C.
- the RNA pellets were washed and dried, then taken up in 50 ⁇ l of H2 ⁇ DEPC.
- the quality of the extraction was checked with 5 ⁇ l of sample on 0.8% agarose gel (TAE 1X). The extracted RNAs were stored at -80 ° C.
- RNA was assayed using a fluorescence spectrophotometer.
- Each extract was diluted to a hundredth in 500 ⁇ l 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 ⁇ g for an absorbance measurement at 260 nm in 1 ml.
- 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
- RNA control the RNA coding for the ribosomal protein L27, the quantity of which did not vary during the experiments
- 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.
- 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.
- 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 ⁇ L 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.
- BSA bovine serum albumin
- 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.
- ITR 3 ′ ITR optimized for the binding of Tnp
- 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 ⁇ l of water to obtain the ITR at a concentration of 50 nM.
- 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 ⁇ g / ml) chloramphenicol (20 ⁇ g / ml ) / 50 mM CaCI 2 overnight (conditions limiting the activity of transposase).
- the culture was titrated on an LB dish (10 ⁇ l of a dilution allowing correct counting), and on an LB-kanamycin dish (100 ⁇ g / ml) (50 ⁇ l of the saturated culture).
- 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.
- 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- ⁇ ⁇ P during the synthesis.
- pET-Tnp 0.5 to 1 ⁇ g of pET-Tnp were incubated for 90 min at 30 ° C with 40 ⁇ l of TNT mix in the presence of ATP- ⁇ 32 P. 5 ⁇ l 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.
- 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).
- D. mauritiana natural host
- P element P element
- a study model in bacteria 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.
- 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.
- transposase mRNAs were absent in bacteria containing the control vector pBadR.
- 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).
- 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.
- 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.
- 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 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 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.
- 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 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).
- transposase 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.
- 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.
- 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).
- transposase produced in the pBadR-Tnp expression system was small but sufficient to have an efficient transposition, even in the absence of an inducer.
- 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).
- this higher production did not translate into a higher transposition frequency compared to the pBadR system.
- 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.
- 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.
- 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.
- 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.
- 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- ⁇ 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.
- 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.
- 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.
- the mutation of the threonine residue at position 88 into a non-phosphorylatable alanine residue 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.
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Abstract
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Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
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US10/543,718 US7732209B2 (en) | 2003-01-28 | 2004-01-23 | Hyperactive, non-phosphorylated, mutant transposases of mariner mobile genetic elements |
CA002514730A CA2514730A1 (fr) | 2003-01-28 | 2004-01-23 | Transposases d'elements genetiques mobiles mariner mutantes, non phosphorylables et hyperactives |
JP2006505659A JP2006518220A (ja) | 2003-01-28 | 2004-01-23 | マリナー可動遺伝因子の突然変異、リン酸化不能および機能亢進性トランスポゼース |
EP04704632A EP1590460A1 (fr) | 2003-01-28 | 2004-01-23 | Transposases d elements genetiques mobiles mariner mutantes, non phosphorylables et hyperactives |
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FR0300905A FR2850395B1 (fr) | 2003-01-28 | 2003-01-28 | Transposases d'elements genetiques mobiles mariner mutantes, non phosphorylables et hyperactives |
FR03/00905 | 2003-01-28 |
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WO2004078981A1 true WO2004078981A1 (fr) | 2004-09-16 |
WO2004078981A8 WO2004078981A8 (fr) | 2005-09-09 |
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PCT/FR2004/000161 WO2004078981A1 (fr) | 2003-01-28 | 2004-01-23 | TRANSPOSASES D'ELEMENTS GENETIQUES MOBILES mariner MUTANTES, NON PHOSPHORYLABLES ET HYPERACTIVES |
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US (1) | US7732209B2 (fr) |
EP (1) | EP1590460A1 (fr) |
JP (1) | JP2006518220A (fr) |
CA (1) | CA2514730A1 (fr) |
FR (1) | FR2850395B1 (fr) |
WO (1) | WO2004078981A1 (fr) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2893951A1 (fr) * | 2005-11-30 | 2007-06-01 | Centre Nat Rech Scient | Procede de production chez les procaryotes de transposases actives et stables d'elements genetiques mobiles mariner |
FR2900935A1 (fr) * | 2006-05-15 | 2007-11-16 | Centre Nat Rech Scient | Systemes de transposition recombinants hyperactifs derives du transposon mos-1 |
WO2009106668A1 (fr) * | 2008-02-29 | 2009-09-03 | Universidad De Jaén | Vecteurs et utilisations du transposon mboumar |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8399643B2 (en) | 2009-02-26 | 2013-03-19 | Transposagen Biopharmaceuticals, Inc. | Nucleic acids encoding hyperactive PiggyBac transposases |
CN103627684B (zh) * | 2013-11-20 | 2016-07-06 | 浙江农林大学 | 人工优化的高活性Mariner-Like转座酶 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2000073510A1 (fr) * | 1999-06-01 | 2000-12-07 | University Of Utah Research Foundation | Procede de mutagenese par transposon dans le nematode caenorhabditis elegans |
WO2001046240A1 (fr) * | 1999-12-22 | 2001-06-28 | Biowindow Gene Development Inc. Shanghai | Nouveau polypeptide, mariner transposase 19 humaine, et polynucleotide codant pour ce polypeptide |
US6368830B1 (en) * | 1999-10-01 | 2002-04-09 | President And Fellows Of Harvard College | Hyperactive mutants of Himar1 transposase and methods for using the same |
-
2003
- 2003-01-28 FR FR0300905A patent/FR2850395B1/fr not_active Expired - Fee Related
-
2004
- 2004-01-23 CA CA002514730A patent/CA2514730A1/fr not_active Abandoned
- 2004-01-23 EP EP04704632A patent/EP1590460A1/fr not_active Withdrawn
- 2004-01-23 WO PCT/FR2004/000161 patent/WO2004078981A1/fr active Application Filing
- 2004-01-23 US US10/543,718 patent/US7732209B2/en not_active Expired - Fee Related
- 2004-01-23 JP JP2006505659A patent/JP2006518220A/ja active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000073510A1 (fr) * | 1999-06-01 | 2000-12-07 | University Of Utah Research Foundation | Procede de mutagenese par transposon dans le nematode caenorhabditis elegans |
US6368830B1 (en) * | 1999-10-01 | 2002-04-09 | President And Fellows Of Harvard College | Hyperactive mutants of Himar1 transposase and methods for using the same |
WO2001046240A1 (fr) * | 1999-12-22 | 2001-06-28 | Biowindow Gene Development Inc. Shanghai | Nouveau polypeptide, mariner transposase 19 humaine, et polynucleotide codant pour ce polypeptide |
Non-Patent Citations (4)
Title |
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AUGE-GOUILLOU C ET AL: "The ITR binding domain of the mariner Mos-1 transposase", MGG MOLECULAR GENETICS AND GENOMICS, vol. 265, no. 1, March 2001 (2001-03-01), pages 58 - 65, XP002256853, ISSN: 1617-4615 * |
DATABASE WPI Section Ch Week 200144, Derwent World Patents Index; Class B04, AN 2001-418030, XP002256854 * |
LOHE ALLAN R ET AL: "Subunit interactions in the mariner transposase", GENETICS, vol. 144, no. 3, 1996, pages 1087 - 1095, XP002256852, ISSN: 0016-6731 * |
ZHANG LEI ET AL: "DNA-binding activity and subunit interaction of the mariner transposase", NUCLEIC ACIDS RESEARCH, vol. 29, no. 17, 1 September 2001 (2001-09-01), pages 3566 - 3575, XP002256851, ISSN: 0305-1048 * |
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2893951A1 (fr) * | 2005-11-30 | 2007-06-01 | Centre Nat Rech Scient | Procede de production chez les procaryotes de transposases actives et stables d'elements genetiques mobiles mariner |
WO2007063033A1 (fr) * | 2005-11-30 | 2007-06-07 | Centre National De La Recherche Scientifique (Cnrs) | Procede de production chez les procaryotes de transposases actives et stables d'elements genetiques mobiles mariner |
JP2009517059A (ja) * | 2005-11-30 | 2009-04-30 | サントル、ナショナール、ド、ラ、ルシェルシュ、シアンティフィク、(セーエヌエルエス) | 原核生物におけるマリナー可動性遺伝因子の活性かつ安定なトランスポゼースの製造方法 |
FR2900935A1 (fr) * | 2006-05-15 | 2007-11-16 | Centre Nat Rech Scient | Systemes de transposition recombinants hyperactifs derives du transposon mos-1 |
WO2007132096A2 (fr) * | 2006-05-15 | 2007-11-22 | Centre National De La Recherche Scientifique (Cnrs) | Systemes de transposition recombinants hyperactifs derives du transposon mos-1 |
WO2007132096A3 (fr) * | 2006-05-15 | 2008-02-14 | Centre Nat Rech Scient | Systemes de transposition recombinants hyperactifs derives du transposon mos-1 |
JP2009537130A (ja) * | 2006-05-15 | 2009-10-29 | サントル、ナショナール、ド、ラ、ルシェルシュ、シアンティフィク、(セーエヌエルエス) | Mos−1トランスポゾンの高活性組換え誘導体の転移のためのシステム |
WO2009106668A1 (fr) * | 2008-02-29 | 2009-09-03 | Universidad De Jaén | Vecteurs et utilisations du transposon mboumar |
ES2334087A1 (es) * | 2008-02-29 | 2010-03-04 | Universidad De Jaen | Vectores y usos del transposon mboumar. |
Also Published As
Publication number | Publication date |
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FR2850395B1 (fr) | 2007-12-14 |
EP1590460A1 (fr) | 2005-11-02 |
US20070031967A1 (en) | 2007-02-08 |
JP2006518220A (ja) | 2006-08-10 |
FR2850395A1 (fr) | 2004-07-30 |
CA2514730A1 (fr) | 2004-09-16 |
WO2004078981A8 (fr) | 2005-09-09 |
US7732209B2 (en) | 2010-06-08 |
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