ES2334087B1 - VECTORS AND USES OF THE TRANSPOSON MBOUMAR. - Google Patents

Abstract

Vectors and uses of the Mboumar transposon.

Production of a naturally active Messor bouvieri transposase recombinantly, genetic constructs derived from the Mboumar transposon necessary for transgenesis and mutagenesis, and uses thereof.

Description

Vectors and uses of the Mboumar transposon.

The present invention is included in the area of molecular biology, specifically within molecular genetics, and more specifically in the field of study of the elements mobile genetics or transposons. Refers to vectors necessary for the construction of genetic tools based in transposons, and their uses.

Prior art

Transposons are DNA sequences that they can move to different positions within the genome of a cell, a process called transposition. In general it classified into two groups:

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Class I transposons or retrotransposons, initially the retrotransposons are copied to themselves to RNA (transcription) but, in addition to transcribing, the RNA is copied into DNA by a reverse transcriptase (often also encoded by the transposon) and inserted back into the genome

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Class II transposons, whose mechanism Transposition does not imply RNA formation. Generally transpose by a process of "cut and paste", by means of a transposase enzyme.

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Transposases are the enzymes responsible for their mobility and are synthesized by the transposon itself. In vivo they can cause transposition of genetic material within the same chromosome, between chromosomes, and even between chromosomes and non-chromosomal genetic material, such as mitochondrial DNA. This movement of genetic material can cause serious disturbances in the organism in which it is produced, although it is also known that it has had great importance throughout evolution. At first the transposons were considered as "junk DNA". However, it is currently thought that they can influence the host genome in different ways. Thus, it is considered that they can modulate gene expression by acting as promoters, activators, silencers, alternative processing sites or sites indicative of epigenetic modifications. They may also contain genes that could be included in the host genome to perform a specific cellular function, a process known as "molecular domestication." In addition to all of the above, due to their existence in multiple copies they can act as recombination sites causing duplications, inversions, deletions or translocations in the genome. (Charlesworth et al . 1994. The evolutionary dynamics of repetitive DNA in eukaryotes. Nature 371: 215-220; Kidwell MG. 2002. Transposable elements and the evolution of genome size in eukaryotes. Genetics 115: 49-63; Ivics & Izsvak 2004 Transposable elements for transgenesis and insertional mutagenesis in vertebrates: a contemporary review of experimental strategies Methods Mol Biol 260: 255-276.) Recent observations show that transposons can be active in somatic cells, opening the possibility that they can play an important role generating diversity between cells with the same genome. Specifically, it has been considered that this diversity could be generated by changes in the regulation of transposase expression which, in turn, could affect the regulation of gene expression of certain genes in certain cells. The effects could be beneficial (as it probably happens in cases of differentiation of neuronal cells, or in the activity of RAG proteins, important in the recombination processes that occur in the lymphocyte genome) or deleterious (carcinogenic processes) depending on the development time and cell type (Collier and Largaespada. Transposable elements and the dynamic somatic genome. Rewied. 2007. Genome Biology 2007, 8 (Suppl 1: S5).

Transposons of the mariner type are sequences capable of moving around the genome that are flanked by inverted terminal repeated sequences ( inverted terminal repeats or ITRs). They encode a single protein, the transposase, that by joining those ITRs cleaves the mariner and integrates it in another place in the genome. The Mos1 transposase catalyzes the movement of the Mos1 transposon found in Drosophila mauritania , which is not only the first isolated mariner element, but also the first naturally active isolated mariner element. The mariner elements constitute a large family of transposons widely spread in the animal kingdom. They encode the enzyme transposase which is the only requirement for its transposition. This enzyme has two domains: the N-terminal domain or DNA binding domain and the C-terminal domain or catalytic domain, which retains the DD (34) D motif. In this enzyme, other reasons and domains essential for its performance have also been identified. Despite the aforementioned, there are still unknown aspects of its performance, whose knowledge could provide great advantages for use as vectors, in addition to providing interesting scientific data; For this reason, in recent years numerous studies are being carried out in this regard.

The wide distribution of mariner elements, also present in humans, and their apparently easy horizontal transfer between different groups of animals, implies that they are capable of functioning in different cellular environments, that is, they do not require specific host factors to lead to Make your transposition. These facts have led to the realization of a series of studies to achieve a widely used vector in animal cells. However, most of the mariner elements that have been cloned so far are defective, since they have undergone mutations in the transposase coding region, so they are unable to produce functional transposases, which has led to transposons currently used as vectors, they are mostly derived from constructions made in the laboratory, using consensus sequences. This seems to be due to a process called "vertical inactivation" (Lohe et al ., 1995. Horizontal transmission, vertical inactivation, and stochastic loss of mariner-like transposable elements. Mol. Biol. Evol . 12: 62 72). Although these elements are naturally inactive (at least with respect to their transposase function), they retain their structure relatively, with several hypotheses in this regard. Thus, the inactive elements may have some regulatory effect, or they may come from the recent introgression of one genome to another.

The importance of using vectors based on transposons in fields as diverse as the study of genes involved in cancer, in the generation of models of human diseases in mice, in pig generation GM with the possibility of use as farms pharmaceuticals, in gene regulation and cell differentiation, in studies of functional genomics etc., has led to the realization of numerous investigations with these vectors used in Different organisms and in different conditions.

In vivo transposition tools have been developed in several organisms. (A summary is presented in Table 1). In most cases, specific hosts required special vectors.

In vivo transposition reactions are carried out independently of the cellular or host conditions: Generally they are carried out with purified transposases, and plasmids, gene fragments, or isolated genomic DNA can be used as targets. In successive reactions, plasmids containing transposons can be transferred to host cells.

In vitro transposition offers many advantages over in vivo systems (Devine and Boeke 1994. Efficient integration of artificial transposons into plasmid targets in vitro : a useful tool for DNA mapping, sequencing and genetic analysis. Nucleic Acids Res , 22: 3765-3772 ; Biery et al . 2000. A simple in vitro Tn7-based transposition system with low target site selectivity for genome and gene analysis. Nucleic Acids Res, 28: 1067-1077), mainly interference with host factors can be avoided. In vitro transposition reactions are generally more efficient and more flexible in choosing DNA targets (Goryshin and Reznikoff. 1998. Tn5 in vitro transposition. J Biol Chem , 273: 7367-7374). Table 2 summarizes the different applications of transposons, both in vitro and in vivo .

The authors of this invention have described an MLE ( mariner-like element ) in the ant Messor bouvieri , of the order Hymenoptera , family Formicidae (Palomeque et al ., Detection of a mariner -like element and a miniature inverted-repeat transposable element (MITE ) associated with the heterochromatin from ants of the genus Messor and their possible involvement for satellite DNA evolution. 2006. Gene 371: 194-205). However, it is not known if this MLE encodes a naturally active transposase, if producing this enzyme recombinantly would retain its possible biological activity, and if the genetic constructs carried out with its elements would be useful for in vitro transposition and / or in vivo

Since most of the transposases found are inactive, and given the large number of applications and interest that MLEs have in biotechnology, it is necessary to construct artificial transposases that are active, from natural defective ones, for use as tools Genetic The most commonly used procedure consists of, from defective copies of the transposases found in various organisms naturally, correct the mutations that inactivate the enzyme by means of directed mutagenesis. Therefore, finding a naturally active transposase , which facilitates its production as a recombinant protein, without the need to introduce modifications to the amino acid sequence, would be a notable advance in this sector of the art.

TABLE 1 Examples of transposable elements useful in transposon applications in different organisms in vivo

one

TABLE 2 Transposon Applications

2

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Explanation of the invention.

The authors of the invention have found that the MLE described in Messor bouvieri encodes the Mboumar transposase, which, produced in the laboratory recombinantly, is the subject of this patent application together with the vectors and other constructions necessary for its use, and is active naturally. As indicated at different points in this application, the remaining active transposases have been reconstructed in the laboratory from defective elements present in the corresponding genomes.

The possibility of having a transposase naturally active, facilitates its use and the construction of new vectors adapted to specific needs of the Biotechnology and its applications.

Thus, the joint use of this transposase and the constructions made with the ITRs can constitute an in vitro transposition system that can be used in various experiments, such as the generation of random mutations. Another possible application of this system is transgenesis and mutagenesis randomly in the genome of prokaryotic or eukaryotic cells, by insertion between the Mboumar ITRs of the desired sequence. The advantage of using vectors based on this type of transposable elements is their versatility, since they are not host-specific, since their only requirement is the presence of the transposase that they themselves produce.

In accordance with a first aspect of the present invention, a recombinant peptide is provided, from now on hereinafter peptide of the invention, consisting essentially of the amino acid sequence SEQ ID NO: 1.

SEQ ID NO: 1 collects the amino acid sequence of the transposase enzyme isolated from the Messor bouvieri species.

When we compare the sequence of the Mboumar transposase (from Messor bouvien ) with the sequence of other transposases isolated in other species, it is clear that there are certain regions where they are more conserved than others, and the same occurs with the transposon. This information may be indicative that these areas are crucial to maintain the structure or function of the protein. Thus, the ITRs of different subfamilies of mariner elements (Himar1, Hpmar1, Hsmar2, Mos1, Csmar1, Ammar1, ..), as well as Mboumar , have different regions conserved (Langin et al ., 1995. The transposable element Impala, a fungal member of the Tc1- mariner superfamily. Mol. Gen. Genet . 246,19-28 (Lampe et al ., 2001. Loss of transposase DNA interaction may underlie the divergence of mariner family transposable elements and the ability of more than one mariner to occupy the same genome Mol. Biol. Evol . 18, 954-961) Both active regions that determine the specificity of transposases and conserved cysteines between which disulfide bridges are established, important for protein conformation, will be more conserved between both proteins, while in other regions the divergence is more apparent, specifically, these enzymes have two domains: the N-terminal domain or DNA binding domain and the C-terminal domain or catalytic domain , which retains the reason DD (34) D (Robertson HM. 1996. Members of the pogo superfamily of DNA-mediated transposons in the human genome. Mol Gen Genet 252: 761-766., Lohe et al . 1997. Mutations in the mariner transposase: the D, D (35) E consensus sequence is non-functional. Proc Natl Acad Sci USA 94: 1293-1297). In this enzyme, other essential motifs and domains for its performance have also been identified (Pietrokovski & Henikoff 1997. A helix-turn-helix DNA-binding motif predicted for transposases of DNA transposons. Mol Gen Genet 254: 689-695., Hickman et al . 2005. Molecular architecture of a eukaryotic DNA transposase. Nat Struct Mol Biol 12: 715-721).

For all the above, it can be expected that the global identity of the transposases homologous to Mboumar , at the level of the amino acids, and more specifically at the level of the amino acid sequence that is collected in SEQ ID NO: 1, is 70% or greater, and more preferably 80% or more and more preferably 90, or 95% or more. The correspondence between the amino acid sequence of the putative transposase (s) and the sequence of other transposases can be determined by methods known in the art. For example, they can be determined by a direct comparison of the amino acid sequence information from the putative homologous transposase, and the amino acid sequence that is collected in SEQ ID NO: 1 of this specification.

In a preferred embodiment of this aspect of the invention, the amino acid sequence of the recombinant peptide has an identity of at least 70% with SEQ ID NO: 1, preferably at least 80%, more preferably at least 90% and even more preferably at least 95%.

Messor bouvieri Mboumar transposase peptides (SEQ ID NO: 1) would maintain their biological function. For example, the transposase activity includes its binding activity to the IRT and / or RDs elements (" Deleted regions "), the cleavage of the nucleic acid sequences between these IRT and / or RDs elements and its sequence insertion activity in A specific target

More preferably, these peptides homologous to the Mboumar transposase have been modified to enhance the biological activity of said enzyme. Thus, for example, amino acids can be substituted in SEQ ID NO: 1 to enhance said activity. Methods for producing such related derived sequences, for example, by site-directed mutagenesis, random mutagenesis, or enzymatic cleavage and / or nucleic acid ligation, are well known in the art, as are the methods for determining whether nucleic acid. thus modified it has a significant homology with the sequence being considered, for example, by hybridization. In another more preferred embodiment of this aspect of the invention the recombinant peptide is homologous to the amino acid sequence of SEQ ID NO: 1.

In another aspect of the invention the peptide Recombinant of the invention is used as a transposase. In other aspect of the invention the recombinant peptide of the invention is Use as a genetic tool.

Another aspect of the invention relates to a nucleic acid sequence or recombinant polynucleotide, of Hereinafter polynucleotide of the invention, which translates to peptide encoded by SEQ ID NO: 1, and that is selected from the list comprising:

to)

nucleic acid molecules that encode a peptide comprising the amino acid sequence of the SEQ ID NO: 1,

b)

nucleic acid molecules whose chain complementary to the polynucleotide sequence of a), or

C)

nucleic acid molecules whose sequence differs from a) and / or b) due to code degeneration genetic.

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In a preferred embodiment of this aspect of the invention the amino acid sequence to which the polynucleotide of the invention has an identity of at least 60% with SEQ ID NO: 1. Preferably, the sequence of amino acids to which the polynucleotide of the invention is translated, it has an identity of at least 80%, more preferably, of 90% and more preferably 95%, with SEQ ID NO: 1.

In another aspect of the invention, the polynucleotide of the invention is used to produce, in amounts so far not available, the peptide of the invention.

Another aspect of the invention relates to a genetic construction, hereafter, first genetic construction of the invention or aid construction, which allows the expression of the peptide of the invention, as it has been described above, in quantities not available until now. Production in quantities greater than currently available would allow the use of the peptide of the invention as genetic tool

Another aspect of the invention relates to a genetic construction, hereafter, second construction genetics of the invention or donor construct, comprising the ITR regions recognized by the peptide of the invention, among the that a polynucleotide of interest is inserted. Additionally, you can contain a marker, such as, but not limited to, an antibiotic resistance gene.

In another aspect of the invention a method for obtaining a recombinant peptide of the invention which consists of putting the described genetic construction above in a suitable reaction medium.

This method includes the cloning vectors and expression comprising the nucleic acid molecules of the invention. Such expression vectors include sequences of adequate control, such as control elements of the translation (such as start and stop codes) and of the transcription (for example, regions of promoter-operator, binding sites. Vectors according to the invention may include plasmids and viruses (comprising bacteriophages and eukaryotic viruses), according to procedures well known and documented in the state of the technique, and can be expressed in a variety of systems of different expression, also well known and documented in the state of the art Suitable viral vectors include baculovirus and also adenovirus and vaccine viruses. Much others viral vectors are also described in the state of the technique.

A variety of techniques are known that can used to introduce such vectors into prokaryotic cells or eukaryotic for expression, or in a germ line or in somatic cells, to form transgenic animals. Techniques suitable transformation or transfection are well described in The bibliography

Eukaryotic host cells or prokaryotic transformed or transfected, containing a nucleic acid molecule according to the invention, as defined previously, they are also part of this aspect of the invention.

Expression of the sequences (same or homologous) encoding the peptide of the invention, using a number of known expression techniques and systems, including expression in prokaryotic cells such as E. coli and in eukaryotic cells such as yeasts or the system of yeasts or baculovirus-insect cell or transformed mammalian cells and in transgenic animals and plants.

In a particular embodiment of this aspect of the invention, as a vector where the Mboumar ORF is introduced, the fusion plasmid pMal-c2X (New England Biolabs) was used, together with the bad E gene of Escherichia coli encoding the protein of maltose binding (MBP: maltose-binding protein ). When the tac promoter of this vector is induced in the presence of IPTG, the Mboumar + MBP transposase fusion protein will be expressed, which will be purified thanks to the affinity that MBP has for amylose and maltose.

To control the insertion of nucleic acid molecules using the Mboumar transposon, dual transposition systems have been developed. A dual system works by contacting at least two genetic constructs (which can be, for example, two plasmids), or by co-transfection or introduction of these into the same cell: the donor and the aid construct. The donor is a gene expression construct (promoter-gene-terminator) or a polynucleotide of interest, flanked by two ITRs (5 'ITR promoter-gene-terminator-3' ITR or 5 'ITR-interest-marker polynucleotide- 3 'ITR or 5' ITR-polynucleotide of interest-3 'ITR) and the aid contains the Mboumar transposase under the control of a promoter. Additionally, the transposition can be carried out massively in vitro , producing the recombinant transposase exogenously, and then putting it in contact with the donor construct.

The contact or co-transfection of both constructs (donor and helper) in the same cell, first causes the expression of the Mboumar transposase and then the Mboumar transposase cuts the donor construction by the 2 ITRs and glue all the construction in the genome of the host cell, or a third recipient genetic construct, which may be, for example, a plasmid (receptor plasmid). In order to cut and paste the ITRs from the donor construct, the Mboumar- mediated transposition requires not only a complete and active Mboumar transposase in the aid construct, but also 2 binding sites for the transposase in the ITRs in the donor construct.

Another aspect of the invention relates to a method for in vitro or in vivo transposition that involves contacting the recombinant peptide of the invention, with the genetic construct comprising the polynucleotide of interest.

In a preferred embodiment of this aspect of the invention the method for in vitro or in vivo transposition is used for the generation of random mutations. In an even more preferred embodiment of the invention, the method for in vitro or in vivo transposition is used for the generation of transgenesis and random mutagenesis in the genome of prokaryotic or eukaryotic cells.

Another aspect of the invention relates to the use of the peptide of the invention for the generation of random mutations, both in vitro and in vivo . More preferably, the peptide of the invention is used for the generation of transgenesis and random mutagenesis in the genome of prokaryotic or eukaryotic cells, both in vitro and in vivo .

Through the insertional mutagenesis induced by the Mboumar transposase, one could identify the function of certain genes, identify yio oncogenes to introduce massive mutagenesis. To increase the number of insertional inactivations, the first ITR constructs inserted into the genome in a first generation can be massively activated by the introduction of exogenous transposases. Inactivation of genes by insertional mutagenesis with ITR would result in obtaining some mutants with the alteration of the desired phenotype. In this process, the inactivated genes are also labeled with the ITRs and their sequences can then be recovered and the genes identified. Once the genes involved have been identified, traditional methods of genetic selection could be used to obtain lines with the desired character.

The term "recombinant peptide that includes "as used herein, refers to to which the peptide includes the amino acid sequence SEQ ID NO: 1, it can integrate with other amino acids and peptides, even fusion peptides, provided that it retains its function as an enzyme transposase, and which is the subject of the present invention. Also does reference to amino acid sequences encoding a peptide counterpart to the one coding for SEQ ID NO: 1.

The term "homology", as it is used in this report, refers to the similarity between two structures due to a common evolutionary ancestry, and more specifically, to the similarity between the amino acids of two or more proteins or amino acid sequences. Since two proteins are considered homologous if they have the same evolutionary origin or if they have similar function and structure, in general, it is assumed that Higher values of similarity or identity of 30% indicate homologous structures. We can consider, therefore, that Identity percentages of at least 80% will maintain the transposase function of said peptide, which is the subject of the present invention.

The term "identity", as used herein, refers to the proportion of identical amino acids between two amino acid sequences that are compared. Sequence comparison methods are known in the state of the art, and include, but are not limited to, the GAG program, including GAP (Devereux et al ., Nucleic Acids Research 12: 287 (1984) Genetics Computer Group University of Wisconsin, Madison, (WI); BLASTP or BLASTN, and FASTA (Altschul et al ., J. Mol. Biol . 215: 403-410 (1999). Additionally, the Smith Waterman algorithm should be used to determine the degree of identity of two sequences.

The term "fragment or derivative" of a peptide, "which possesses transposase activity" refers to fragments or peptides derived from the Mboumar transposase in their natural state that lack some or some amino acids, and which still act by transposing DNA. Alternatively, this term also refers to peptides derived from the natural Mboumar transposase, where one or more amino acids have been changed, removed, added, or less preferably, which has undergone reversals or duplications. Such modifications are preferably made by recombinant DNA technology. Other modifications can also be made by chemical alterations of the transposase. The resulting peptide (or fragments derived therefrom) can be produced recombinantly, and retain identical, or essentially identical, characteristics to the natural Mboumar transposase.

A "recombinant peptide" as it is understood in this report, is one that is obtained from the expression of a "recombinant polynucleotide".

The term "recombinant polynucleotide" as used herein assumes a polynucleotide of genomic origin, CDNA, semi-synthetic, or synthetic that, by virtue of its origin or handling: (1) is not associated with the total or a portion of a polynucleotide with which it is associated in nature, (2) is bound to a polynucleotide different from that to which it is bound in the  nature, or (3) does not occur in nature.

The term "polynucleotide" as here is usa refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, this term includes double or single stranded DNA, just like that double or single stranded RNA. It also includes all types of known modifications (markers known in the art, methylation, auctions, replacement of one or more of the nucleotides natural with an analog, internucleotide modifications such as, by for example, those with unloaded joints (for example, phosphonates of methyl, phosphorus tri esters, phosphorus amidates, carbamates, etc.) and with loaded joints (for example, phosphorus thioates, phosphorus dithioates, etc.), those containing halves pendants, such as proteins (including, for example, nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalants (for example, acridine, psoralen, etc.), those that contain chelators (e.g. metals, radioactive metals, boron, oxidative metals, etc.), those that contain alkylating agents, those with modified unions (for example, acids anomeric alpha nucleic, etc.), as well as unmodified forms  of the polynucleotide.

The term "genetic tool" as defined herein, refers to the tools used in genetic engineering for various applications, and includes tools of molecular genetics, quantitative genetics, population genetics, etc ... In particular, in the context of the present invention, it refers to a tool useful for in vitro transposition, or for transgenesis and mutagenesis randomly, both in vitro and in vivo .

A "vector" is a replicon that has been joined another polynucleotide segment, to perform replication and / or expression of the joined segment.

A "replicon" is any element genetic that behaves as an autonomous unit of replication polynucleotide within a cell; that is, able to replicate under his own control.

"Control sequence" refers to polynucleotide sequences that are necessary to effect the expression of the coding sequences to which they are linked. The nature of such control sequences differs depending of the host organism; in prokaryotes, said control sequences they generally include a promoter, a ribosomal binding site, and termination signals; in eukaryotes, generally, said control sequences include promoters, termination signals, intensifiers and, sometimes, silencers. It is intended that the term "control sequences" include, at a minimum, all the components whose presence is necessary for expression, and it can also include additional components whose presence is advantageous

"United operatively" refers to a juxtaposition in which the components thus described have a relationship that allows them to function in the intended way. A control sequence "operatively linked" to a coding sequence is linked in such a way that the expression of the coding sequence is achieved under compatible conditions With the control sequences.

A "free reading frame" (ORF) is a region of a polynucleotide sequence that encodes a polypeptide; this region can represent a portion of a coding sequence or a complete coding sequence.

A "coding sequence" is a polynucleotide sequence that is transcribed to mRNA and / or is translates to a polypeptide when under sequence control appropriate regulators. The limits of the coding sequence are determined by a translation start codon at the end 5 'and a translation completion codon at the 3' end. A  coding sequence may include, but is not limited to mRNA, CDNA, and recombinant polynucleotide sequences.

The term "natural Mboumar ", as used herein, refers to the peptide or protein encoded by the nucleotide sequence called Mboumar-9, corresponding to SEQ ID NO: 1, cloned from the Messor ant genome bouvieri .

The term "mutant Mboumar " means in this description, the protein resulting from the modification of the "natural Mboumar " protein, preferably to enhance the biological activity of said protein.

A "host" or "host cell" as used herein refers to an organism, cell or tissue that serves as a target or recipient of the transposable element to insert themselves. A host cell or host may also indicate a cell or host that expresses a recombinant protein of interest where the cell host is transformed with an expression vector containing the gene of interest

The term "transgenic" is used in the context of the present invention to describe animals or plants in which a non-proprietary DNA sequence introduced by a mariner element (mariner-like element MLE) has been stably incorporated into its chromosomes of such that it can pass stably to successive generations of transgenic descendants. In such circumstances, the initial transgenic organism is known as the "founder." The "founder" animal must have the non-own DNA or transgene incorporated in all its cells or in a proportion sufficient for its progeny to establish by inheritance the transgenic. When the transgene is present only in a portion of cells, the organism is referred to as a chimera. The present invention also extends to animals that incorporate the transgene stable or directly into their chromosomes and which express the transgene in their somatic cells without passing the transgene to their offspring. Specifically, these animals would serve as genetic models, for, for example, but not limited to these uses, to identify and study the function of genes, detect oncogenes, obtain biopharmaceuticals, ... Similar models described by genetic constructs derived from transposons in mice and zebrafish.

It is understood by "transgenesis" in this memory to the process of transferring non-own DNA to an organism, which This happens to be called "transgenic".

Throughout the description and the claims the word "comprises" and its variants not they intend to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects,  advantages and features of the invention will be partly detached of the description and in part of the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention.

Brief description of the figures

Figure 1. Plasmid pITR and plasmid pITR-Kn. The pITR vector is obtained from the plasmid donated with the Mboumar-9 pGEMT easy vector mariner (Promega) by eliminating the ampicillin resistance gene (Ampr) and the origin of replication (ori). The EcoRI targets of the pGEMT easy vector have also been removed.

Figure 2. Donation site of plasmid pITR with a region of cut of Apo I.

Figure 3. Procedure for removing EcoRI targets from pGEMT easy vector. A- Digestion with E. coli and generation of protruding ends. B- Filling the protruding ends with Klenow and generating blunt ends. C- Ligating of the two ends with T4 ligase.

Figure 4. Scheme of the in vitro transposition assay .

Detailed statement of embodiments

Several different copies of mariner elements have been donated in the ant Messor bouvieri . The analysis of the nucleotide sequence of these mariner indicates that they could give rise to functional transposases, since the transposase coding region does not present mutations that generate stop codons, and the protein it would originate retains most of the reasons that are assumed they are necessary for transposase activity.

From one of the mariner clones obtained (Mboumar-9) the transposase coding region has been isolated, which has been introduced into an expression vector to carry out its synthesis in E. coli . This transposase has been called Mboumar . Through in vitro transposition experiments it has been determined that this transposase is active, being able to mobilize the mariner element from which it comes.

Several constructions have been carried out by partially eliminating the central region of the mariner , maintaining the ITRs. The deleted region has been replaced by a marker gene, which in this case is the kanamycin resistance gene (Kn). Through in vitro transposition it has been proven that this construct (ITR-Kn-ITR gene) can be mobilized by the Mboumar transposase, and, therefore, any other sequence placed between these ITRs.

The invention will now be illustrated by tests carried out by the inventors, which shows the activity of the recombinant peptide of the invention and the effectiveness of the genetic constructs and of said peptide in in vitro transgenesis.

a) Mboumar transposase purification

In vitro production of the protein is carried out by introducing its coding region (ORF) into an expression vector. Of the multiple expression vectors available on the market, the fusion plasmid pMal-c2X (New England Biolabs) has been used in this case.

The Mboumar ORF is introduced into the pMal-c2X vector downstream of the Escherichia coli malE gene that encodes maltose binding protein (MBP: maltose-binding protein). When the tac promoter of this vector is induced in the presence of IPTG, the Mboumar + MBP transposase fusion protein will be expressed, which will be purified thanks to the affinity that MBP has for amylose and maltose.

The Mboumar transposon ORF is amplified from the Mboumar -9 clone (Palomeque et al . 2006. Detection of a mariner -like element and a miniature inverted-repeat transposable element (MITE) associated with the heterochromatin from ants of the genus Messor and their possible involvement for satellite DNA evolution (Gene 371: 194-205) with two primers that incorporate two cutting targets for the restriction enzymes EcoRI and BamHI, which will allow it to be introduced in phase in the polylinker of pMal-c2X. Once the plasmid that includes the Mboumar ORF is obtained at the correct donation site in pMal-c2X, it is introduced by transformation into E. coli bacteria of the Rosetta 2 strain. With one of the positive colonies obtained, 100 ml of Liquid culture of LB medium with ampicillin (Amp), and incubated with shaking at 37 ° C until an A = 0.5 is obtained. IPTG is then added to a final concentration of 0.1 mM and kept under stirring overnight at room temperature. The culture is then centrifuged at 5,000 rpm for 10 minutes. The precipitate is resuspended in 10 ml of HSG buffer (Hepes pH = 7.5 50 mM, 200 mM NaCl, 5 mM EDTA, 2 mM DTT and 10% glycerol) to which protease inhibitor is added. The 100 ml of culture are introduced in a French press and the bacteria are subjected to a pressure of 1,000 Psi. The lysate obtained is centrifuged at 15,000 rpm for 30 minutes. The supernatant is passed through a column with 1 ml of amylase (previously washed with Buffer HSG) where the fusion protein will be retained. Once all the supernatant has passed through the amylose matrix, it is washed again with Buffer HSG. Finally, to separate the protein, 1 ml of Buffer HSG with maltose is added at a concentration of 10 mM, diluted in buffer A (Hepes pH = 7.5 50 mM, 50 mM NaCl, 5 mM EDTA, 2 mM DTT and a 5% glycerol) and is passed through an ion exchange column (FPLC) taking several fractions. These fractions are migrated on an SDS-Page gel and the one with a higher proportion of complete protein than the incomplete one is selected. To the selected fraction is added glycerol (final concentration of 10%), aliquot and stored at -80 ° C for later use. The protein concentration is quantified by the Bradford method.

The sequence of the transposase Mboumar -9, obtained from the DNA sequence of marinating Mboumar-9, is included in SEQ ID NO: 1.

b) Basis of in vitro transposition

To determine the in vitro activity of the transposase it is necessary to develop two vectors, one the donor and the other the receptor. Donor plasmid contain the sequence desired to mobilize, which must be flanked by ITRs Mboumar transposon. The Receptor Plasmid can be any other where the insertion of the sequence located between the two ITRs provided by the donor plasmid can be detected.

During the in vitro transposition the Mboumar transposase will recognize the ITRs that are located in the donor plasmid. The transposase will make a cut at the ends of these ITRs and integrate this DNA fragment into a TA dinucleotide (which will undergo duplication during the process) of the recipient plasmid. However, it is also possible that integration takes place at a site other than the donor plasmid itself. To avoid this inconvenience, the donor plasmid must be unable to replicate in the bacteria that will be transformed with the in vitro transposition reaction.

c) Design of vectors for in vitro transposition

Two donor plasmids have been constructed: pITR, which presents the ends of the mariner element and therefore its ITRs, and the pITR-Kn, derived from the previous one, but to which the kanamycin resistance gene (Knr gene) has been incorporated ( Fig. 1).

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c.1. PITR plasmid

For the construction of this vector we start from the Mboumar -9 plasmid that contains the Mboumar -9 mariner element cloned in the plasmid pGEMT easy vector (Promega). The first modification that is made on the plasmid is the elimination of the ampicillin resistance gene (Ampr) and the origin of replication (ori). For this, the plasmid has been digested with the enzymes Scal and Accl, thus releasing most of the Ampr gene and the ori. This fragment is replaced by the origin of R6K replication, which will allow this plasmid to replicate only in pir + bacteria, but not in pir- strains, which will be those transformed with the transposition reaction.

Subsequently, the plasmid is digested with the restriction enzymes Mscl and Ndel (partial digestion), and the ends generated in the vector are joined by ligation. These enzymes will release the central region of the mariner leaving only 173 bp of the ends, where the two ITRs are found. In this region a target sequence for the Apol enzyme is located, which can be used to insert a DNA sequence (Fig. 2). However, this enzyme is also able to cut on the EcoRI targets found in the pGEM-T vector, on both sides of the insert, so it is necessary to eliminate them.

To eliminate the EcoRI targets, the plasmid is digested with this enzyme, generating two fragments, one with the insert corresponding to the mariner and the other with the vector. This enzyme generates protruding ends that are filled with the help of the Klenow enzyme, thus generating fragments with blunt ends that are re-bound, thus degenerating the target for EcoRl (Fig. 3), leaving the Apol target between the two ITRs as the single target for this enzyme in the plasmid (Fig. 2)

c.2. PITR-Kn plasmid

Plasmid pITR-Kn is derived of the pITR to which the resistance gene has been inserted Kanamycin (Knr gene) on Apol's target (Fig. 1)

c.3. Receptor plasmid

As a receptor plasmid, any other that confers resistance to an antibiotic other than kanamycin and is capable of replicating in pyr- bacteria can be used. For the in vitro transposition assays the pGEMT Easy Vector (Promega) vector itself has been used.

d) In vitro transposition test (Figure 4)

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\ vtcortauna Elements necessary for in vitro transposition:

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\ vtcortauna Donor plasmid (pITR-Kn).

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\ vtcortauna Plasmid receptor (pGEM-T).

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\ vtcortauna Purified Mboumar transposase.

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\ vtcortauna Reaction buffer: It is the buffer in which the necessary conditions are met for the transposition by the Mboumar transposase.

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\ vtcortauna In vitro transposition reaction:

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\ vtcortauna Donor plasmid: 9 µM.

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\ vtcortauna Receiving plasmid: 9 µM.

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\ vtcortauna Mboumar transposase: 10 nM.

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\ vtcortauna 1x reaction buffer:

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\ vtcortauna Glycerol: 10%.

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\ vtcortauna Hepes (pH = 7.9): 25 mM.

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\ vtcortauna BSA: 12, 5 \ mug / \ mul.

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\ vtcortauna DTT: 2 mM.

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\ vtcortauna NaCl: 100 mM.

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\ vtcortauna MgCl2: 10 mM.

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This reaction is incubated at 28 ° C throughout the night. During this period the transposase cleaves the Knr gene together to the ITRs that flank it and integrate it into TA dinucleotide of the recipient plasmid (or donor plasmid). The mobilization of this sequence to the recipient plasmid will generate a plasmid that will confer resistance to kanamycin and ampicillin, and that will be able to replicate in pir bacteria.

Next bacteria are transformed competent pir- (DH5α) with 8 µl of the reaction of transposition and negative control (without transposase). Half of the volume of the transformation with the transposition reaction is  plating on LB plates with kanamycin and ampicillin, and the other half on plates with only ampicillin. The same volume is the one that will sow the transformation with the negative control in a LB plate + ampicillin + kanamycin.

On plates with only ampicillin they can grow all those bacteria that have incorporated the receptor plasmid original or with the insertion of the Kn r gene, since the plasmid receptor contains the Amp r gene.

In the plates with ampicillin and kanamycin only those bacteria that possess the receptor plasmid with the insertion of the Kn r gene can grow, as long as the integration has occurred outside the Amp r gene and the origin of replication ( ori ). Integration at any of these sites would result in non-viable bacteria, since integration in the Amp r gene would make the bacteria not resistant to ampicillin, and integration into the ori would prevent replication of the recipient plasmid within of the bacteria

Bacteria that have incorporated the donor plasmid (with or without the Kn R gene) will not survive, since this plasmid is not able to replicate in the pir- DH5? Bacteria.

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\ vtcortauna Transposition Efficiency Calculation:

The efficiency with which the Mboumar transposase has carried out the transposition under the conditions described is 10-3 . This is calculated by dividing the number of Transformer Plasmids transformed with the Kn R gene integrated by the total number of Transformed Receptor Plasmids.

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4

Claims (16)

1. Recombinant peptide comprising the amino acid sequence SEQ ID NO: 1. 2. Recombinant peptide according to claim 1 whose amino acid sequence has at least one identity 70% with SEQ ID NO: 1. 3. Recombinant peptide according to claim 1 that has an identity of at least 80% with the sequence of amino acids of SEQ ID NO: 1. 4. Recombinant peptide according to claim 1 that has an identity of at least 90% with the sequence of amino acids of SEQ ID NO: 1. 5. Recombinant peptide according to claim 1 that has an identity of at least 95% with the sequence of amino acids of SEQ ID NO: 1. 6. Peptide homologous recombinant peptide according to any of claims 1-5. 7. Isolated polynucleotide, which translates to the amino acid sequence of SEQ ID NO: 1, and is selected from the list comprising:

to.

nucleic acid molecules that encode a polypeptide comprising the amino acid sequence of SEQ ID NO: 1,

b.

nucleic acid molecules whose chain complementary to the polynucleotide sequence of a), or

C.

nucleic acid molecules whose sequence differs from a) and / or b) due to code degeneration genetic.

for use in the production of recombinant peptide according to any of the claims 1-6.

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8. Polynucleotide according to claim 7, where the amino acid sequence to which it is translated presents a Identity of at least 70% with SEQ ID NO: 1. 9. Polynucleotide according to claim 7, where the amino acid sequence to which it is translated presents a Identity of at least 80% with SEQ ID NO: 1. 10. Polynucleotide according to claim 7, where the amino acid sequence to which it is translated presents a Identity of at least 90% with SEQ ID NO: 1. 11. Polynucleotide according to claim 7, where the amino acid sequence to which it is translated presents a Identity of at least 95% with SEQ ID NO: 1. 12. Polynucleotide according to any of the claims 7-11, encoding a peptide according to any of claims 1-6. 13. Use of the recombinant peptide, according to any of claims 1-6, or a fragment or derivative thereof, as transposase. 14. Use of the recombinant peptide, according to any of claims 1-6, as a useful tool for transposition in vitro , or for transgenesis and mutagenesis randomly, both in vitro and in vivo . 15. Use of the recombinant peptide according to claim 14, for the generation of random mutations in vitro . 16. Use of the recombinant peptide according to claim 14, for the generation of transgenesis and random mutagenesis in vitro , in the genome of prokaryotic or eukaryotic cells.