FR2853906A1 - New polypeptides from Lactobacillus with N-deoxyribosyltransferase activity, useful for preparation of deoxyribonucleotide analogs, e.g. antiviral and antitumor agents, also new nucleic acids encoding them - Google Patents

Abstract

Isolated and purified polypeptides (A) from Lactobacillus has at least one N-deoxyribosyltransferase activity and is any of 5 sequences reproduced; (4), (6), (8), (10) and (12) with, respectively, 167, 168, 84, 133 and 159 amino acids (aa). Independent claims are also included for the following: (1) isolated polypeptides (A1) that contain (a) the new sequences, (b) their variants or homologs with at least 80% identity, (c) their fragments of at least 15 aa, or (d) a biologically active fragment of (a) or (b); (2) purified and isolated polynucleotides (B) that encode (A) or (A1); (3) isolated polynucleotides (B1) that are (a) any of sequences (3; 504 bp), (5; 516 bp), (7; 255 bp), (9; 399 bp) or (11; 480 bp), (b) a fragment of at least 15 consecutive nt from (a), (c) a sequence at least 70% identical to (a) or (b), or (d) the complement or RNA corresponding to (a)-(c); (4) recombinant cloning and/or expression vector containing (B) or (B1), or expressing (A) or (A1); (5) host cell transfected with the vector of (4); (6) non-human organisms (metazoal animals or plants) that contain a cell of (5); (7) producing recombinant polypeptides by culturing cells of (5); (8) recombinant polypeptide (A2) produced by method (7); (9) mono- or poly-clonal antibodies (Ab), or their fragments, that bind selectively to (A)-(A2); and (10) process for in vitro or in vivo enzymatic synthesis of deoxyribonucleotides that includes at least one step catalyzed by (A) or (A1). ACTIVITY : Antibacterial; Virucide; Anti-HIV; Antiparasitic; Fungicide; Cytostatic; Herbicide; Insecticide. No biological data given. MECHANISM OF ACTION : None given.

Description

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 The present invention relates to the field of biology, and more particularly to the microbiological production of base analogs. The present invention relates to novel polypeptides and fragments thereof, isolated from Lactobacillus, having at least one N-deoxyribosyltransferase activity, polynucleotides encoding said polypeptides, cloning and / or expression vectors including said polynucleotides, cells transformed by said vectors. and specific antibodies directed against said polypeptides. The invention also relates to a process for the enzymatic synthesis of deoxyribonucleosides.

 Nucleoside analogues whose structure involves alterations of the sugar or of the heterocyclic base, form a family of molecules active in the treatment of numerous bacterial, viral, parasitic and fungal infections as well as in antitumor chemotherapy [Périgaud et al, 1992] . In addition, the insecticidal and herbicidal properties of certain nucleoside antibiotics make them potential agents in the agrochemical and environmental sector [Isono, 1988].

The industry uses two modes of production of these analogs, organic synthesis and biocatalytic conversion (enzymatic conversion and microbiological conversion), which have opposite advantages and disadvantages. Organic synthesis provides access to the most varied chemical structures but requires multiple and expensive steps in reagents and solvents. On the contrary, biocatalytic methods allow easy production in an aqueous medium but limited to a small number of compounds

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 possible because of the specificity of the enzymes, which admit a limited range of analogues in place of their physiological substrates. Nucleosides phosphorylase and N-deoxyribosyltransferase, which are derived from purine and pyrimidine rescue pathways in bacteria, are the most used enzymes for these enzymatic conversions (Krenisky et al, 1981).

 There is therefore an urgent need to obtain nucleoside-converting enzymes and their derivatives, having an enlarged enzymatic activity in order to diversify the industrial production of these compounds.

This is the technical problem that the inventors of the present invention proposes to solve.

 The N-deoxyribosyltransferase of Lactobacillus leichmannii as well as that of L. helveticus, partially purified or not, appears to be the best donor of glycosyl group and tolerates a large number of structural variations on the basis.

This enzyme has been used to produce a number of analogs including 2-chloro, 2'-deoxyadenosine (Carson et al, 1984), the 2 ', 3'-dideoxynucleosides of natural bases (Carson et al. Wasson, 1988) or several pyrazolo (3,4-d) pyrimidine and triazolo (4,5-d) pyrimidine derivatives of 2 ', 3'-dideoxycytidine and the corresponding base (Fischer et al, 1990).

 In order to have recombinant enzymes capable of treating the widest variety of deviating substrates either by the base or by the sugar, the inventors have isolated genes coding for an N-deoxyribosyltransferase activity of different strains of

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 lactobacilli. This variety of Ndeoxyribosyltransferase enzymes makes it possible to increase the chances of obtaining enzymes whose specificity is broadened by mutations in wild-type genes or by chimera of these wild-type genes.

t997 ; dbjBAA92683.2(-8039914)). Two classes of N-deoxyribosyltransferase have been distinguished (Danzin and Cardinaud, 1976), the first (class I) designated ptd (for purine transdesoxyribosylase) exclusively catalyzing the exchange of deoxyribose between two purines: dR-Pur + Pur '<-> dR-Pur '+ Pur and the second (class II) - designated ntd (for nucleoside transdesoxyribosylase), the exchange of deoxyribose between a purine and a pyrimidine, between two pyrimidines or between two purines: dR-Pyr + Pur <-> dR-Pur + Pyr dR-Pyr + Pyr '<->dR-Pyr' + Pyr dR-Pur + Pur '<->dR-Pur' + Pur
Only two genes specifying class II enzymes, designated ntd, have been reported to date (Hück,

t997; dbjBAA92683.2 (-8039914)).

 The subject of the present invention is therefore an isolated or purified polypeptide of Lactobacillus having at least one N-deoxyribosyltransferase activity of amino acid sequence selected from the sequences SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14.

 According to a preferred embodiment, the polypeptide according to the invention is the N deoxyribosyltransferase of SEQ ID No. 2 (or SEQ ID No. 14) encoded by the ntd Lh gene of Lactobacillus helveticus.

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 According to a second preferred embodiment, the polypeptide according to the invention is the N deoxyribosyltransferase of SEQ ID No. 4 encoded by the ptd Lh gene of Lactobacillus helveticus.

 According to a third preferred embodiment, the polypeptide according to the invention is the N deoxyribosyltransferase of SEQ ID No. 6 coded by the ntd Lf gene of Lactobacillus fermentum.

 According to a fourth preferred embodiment, the polypeptide according to the invention is the N deoxyribosyltransferase of SEQ ID No. 8 encoded by the ntd gene of Lactobacillus crispatus.

 According to a fifth preferred embodiment, the polypeptide according to the invention is the N deoxyribosyltransferase of SEQ ID No. 10 encoded by the ntd gene of Lactobacillus amylovorus.

 According to a sixth preferred embodiment, the polypeptide according to the invention is the N deoxyribosyltransferase of SEQ ID No. 12 encoded by the ntd gene of Lactobacillus acidophilus.

 The isolated polypeptide according to the invention is characterized in that it comprises a polypeptide chosen from (a) a polypeptide of sequence SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10. , SEQ ID NO: 12, SEQ ID NO: 14; (b) a polypeptide variant polypeptide of amino acid sequences defined in a); (c) a polypeptide homologous to the polypeptide defined in (a) or (b) and having at least 80% identity, preferably 85%, 87%, 90%, 95%, 97% 98%, 99% of identity with said polypeptide of a); (d) a fragment of at least 15 consecutive amino acids preferably 17,20, 23,25, 30, 40,50, 100,250 amino-

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 consecutive acids of a polypeptide defined in a), b) or c); and (e) a biologically active fragment of a polypeptide defined in a), b) or c).

 The polypeptide according to the invention is characterized in that it makes it possible to satisfy the guanine requirement of certain bacterial strains such as PAK6 which is a strain of E. coli. coli whose two genes of the guaBA operon, which control the conversion of IMP to XMP then into GMP, as well as those of the deoCABD operon that control the deoxynucleoside degradation, have been deleted. Indeed, these strains to survive or grow require the addition of deoxyguanosine (dR-Q) in the culture medium and the presence of an N deoxyribosyltransferase activity of a polypeptide according to the invention to carry out the exchange: + A < -> dR-A + G.

 In the present description, the term "polypeptide" will also be used to designate a protein or a peptide.

 The term "variant polypeptide" will be understood to mean all the mutated polypeptides that can exist naturally, in particular in humans, and that correspond in particular to truncations, substitutions, deletions and / or additions of amino acid residues.

 The term "homologous polypeptide" will be understood to denote polypeptides having, relative to the natural deoxyribosyltransferases of Lactobacillus according to the invention, certain modifications such as in particular a deletion, addition or substitution of at least one amino acid, a truncation, an elongation and / or a chimeric fusion. Among the homologous polypeptides, those whose acid sequence is preferred are

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 The amino acids have at least 80% identity, preferably at least 85%, 87%, 90%, 93%, 95%, 97%, 98%, 99% identity with the amino acid sequences of the amino acids. polypeptides according to the invention. In the case of a substitution, one or more consecutive or non-consecutive amino acids may be replaced by equivalent amino acids. The term "amino acid equivalent" is intended herein to designate any amino acid that may be substituted for one of the amino acids of the basic structure without, however, modifying the essential functional characteristics or properties, such as their biological activities (ie deoxyribosyltransferase), corresponding polypeptides such as the in vivo induction of antibodies capable of recognizing the polypeptide whose amino acid sequence is included in the amino acid sequence SEQ ID N 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14, or one of its fragments. These equivalent amino acids can be determined either on the basis of their structural homology with the amino acids to which they are substituted, or on the results of the cross-biological activity tests to which the various polypeptides are likely to give rise. By way of example, mention may be made of the possibilities of substitutions that may be carried out without resulting in a thorough modification of the biological activities of the corresponding modified polypeptides, the replacements, for example, of leucine by valine or isoleucine. , aspartic acid with glutamic acid, glutamine with asparagine, arginine with lysine etc., substitutions

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 inverses being naturally conceivable under the same conditions.

 By polypeptide fragment is meant a polypeptide comprising at least 15 consecutive amino acids, preferably 17,20, 23,25, 30,40, 50, 100,250 consecutive amino acids. The polypeptide fragments according to the invention obtained by cleavage of said polypeptide by a proteolytic enzyme, by a chemical reagent, or by placing said polypeptide in a highly acidic environment also form part of the invention.

 By biologically-active fragment, will be meant in particular a polypeptide amino acid sequence fragment according to the invention having at least one of the characteristics or functional properties of the polypeptide according to the invention, especially in that it comprises an activity N-deoxyribosyltransferase. The variant polypeptide, the homologous polypeptide or the polypeptide fragment according to the invention has at least 10%, preferably 20%, 30, 40%, 50%, 60%, 70%, 80%, 90%, 95% of the polypeptide. N-deoxyribosyltransferase activity.

 Various protocols known to those skilled in the art have been described for introducing mutations into polypeptides. Among these, it is worth mentioning as an example the polymerase chain reaction (PCR) in the presence of manganese (Cadwell et al., 1992). The mutations can be introduced either randomly - in this case the mutagenesis step is followed by a step of screening the mutant of interest - either in a targeted manner. In the latter case, mutations are

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 preferably introduced at the catalytic site N-deoxyribosyltransferases according to the invention.

 Preferably a polypeptide according to the invention is a polypeptide consisting of the sequence SEQ ID N 2, SEQ ID N 4, SEQ ID N 6, SEQ ID N 8, SEQ ID N 10, SEQ ID N 12, SEQ ID N 14 or a sequence having at least 80% identity, preferably at least 85%, 90%, 95%, 98% and 99% identity with SEQ ID N 2, SEQ ID N 4, SEQ ID N 6 , SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 after optimal alignment. A polypeptide whose amino acid sequence has an identity percentage of at least 80%, preferably at least 85%, 90%, 95%, 98% and 99% after optimal alignment with a reference sequence is meant polypeptides having certain modifications relative to the reference polypeptide, such as in particular one or more deletions, truncations, an elongation, a chimeric fusion, and / or one or more substitutions.

 Among the polypeptides whose amino acid sequence has an identity percentage of at least 80%, preferably at least 85%, 90%, 95%, 98% and 99% after optimal alignment with the SEQ sequences ID NO 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, SEQ ID NO 10, SEQ ID NO 12, SEQ ID NO 14 or with one of their fragments according to the invention, polypeptides are preferred. variants encoded by the variant peptide sequences as defined above, in particular the polypeptides whose amino acid sequence exhibits at least one mutation corresponding in particular to a truncation, deletion, substitution and / or addition of at least one acid residue amine compared to SEQ ID sequences

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 N 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 or with one of their fragments; more preferably, the variant polypeptides having at least one mutation which decreases the specificity of the polypeptide according to the invention for its substrate, so that the variant polypeptides according to the invention are capable of catalyzing a wider variety of substrate, in order to to obtain a wider range of basic analogues.

 The invention also relates to a purified or isolated polynucleotide characterized in that it encodes a polypeptide as defined above, and preferably for a polypeptide of sequence SEQ ID N 2, SEQ ID N 4, SEQ ID N 6, SEQ ID N 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14. Preferably, the polynucleotide according to the invention has the sequence SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7. , SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13.

 The isolated or purified polynucleotide according to the invention is characterized in that it comprises a polynucleotide chosen from (a) SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ. ID N 11, SEQ ID N 13; (b) the sequence of a fragment of at least 15 consecutive nucleotides, preferably at least 18, 21,24, 27,30, 35,40, 50, 75,100, 150, 200 consecutive nucleotides of the sequence SEQ ID N 1, SEQ ID NO 3, SEQ ID NO 5, SEQ ID NO 7, SEQ ID NO 9, SEQ ID NO 11, SEQ ID NO 13; (c) a nucleic sequence having an identity percentage of at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, 98% and 99% after optimal alignment with a sequence defined in a) or b); (d) the complementary sequence or the RNA sequence

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 corresponding to a sequence as defined in a), b) or c).

The polynucleotide according to the invention is also characterized in that its expression in host cells, in particular bacterial strains such as PAK6, makes it possible to satisfy the guanine requirement of said strain. The PAK6 strain was deposited at the CNCM on May 2, 2001 under the N 1-2664. The strain PAK6 corresponds to the bacterial strain of Escherichia coli MG 1655 deleted from two genes guaA and guaB as well as genes deoC, deoA, deoB, deoD. The strain PAK6 is auxotrophic for guanine in minimal glucose medium.
By nucleic acid, nucleic or nucleic acid sequence, polynucleotide, oligonucleotide, polynucleotide sequence, nucleotide sequence, terms which will be used indifferently in the present description, is meant to designate a precise sequence of nucleotides, modified or otherwise, to define a fragment or a region of a nucleic acid, with or without unnatural nucleotides, and which may correspond to both double-stranded DNA, single-stranded DNA and transcripts of said DNA, and / or an RNA fragment.

 It should be understood that the present invention does not relate to nucleotide sequences in their natural chromosomal environment, i.e., in the natural state. These are sequences that have been isolated and / or purified, that is to say that they were taken directly or indirectly, for example by copy, their environment having been at least partially modified. We also hear

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 designate the nucleic acids obtained by chemical synthesis.

 By polynucleotide of complementary sequence, is meant any DNA whose nucleotides are complementary to those of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13 or a part of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13 and whose orientation. is reversed.

 By percentage identity between two nucleic acid or amino acid sequences in the sense of the present invention, it is meant to designate a percentage of nucleotides or identical amino acid residues between the two sequences to be compared, obtained after the best alignment, this percentage being purely statistical and the differences between the two sequences being randomly distributed over their entire length. The term "better alignment" or "significant alignment" is understood to mean the alignment for which the percentage of identity determined as hereinafter is the highest.

Comparisons of sequences between two nucleic acid or amino acid sequences are traditionally performed by comparing these sequences after having significantly aligned them, said comparison being carried out by segment or by comparison window to identify and compare the local regions of sequence similarity.

The significant alignment of the sequences for the comparison can be realized, besides manually, by means of the local homology algorithm of Smith and Waterman (1981), by means of the algorithm of local homology of Neddleman and Wunsch (1970) ), by means of

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 similarity search method of Pearson and Lipman (1988), using computer software using these algorithms (GAP, BESTFIT, BLAST P, BLAST N, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr. , Madison, WI).

In order to obtain significant alignment, the BLAST program is preferably used with the BLOSUM 62 matrix. The PAM or PAM250 matrices can also be used.

 The percentage identity between two nucleic acid or amino acid sequences is determined by comparing these two significantly aligned sequences, the nucleic acid or amino acid sequence to be compared being able to include additions or deletions with respect to the reference sequence for a significant alignment between these two sequences. The percent identity is calculated by determining the number of identical positions for which the nucleotide or amino acid residue is identical between the two sequences, dividing this number of identical positions by the total number of positions compared and multiplying the number of identical positions. result obtained by 100 to obtain the percentage of identity between these two sequences.

 By nucleic sequences having an identity percentage of at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, 98% and 99% after significant alignment with a reference sequence is meant nucleic sequences having, with respect to the reference nucleic sequence, certain modifications such as deletion, truncation, elongation, chimeric fusion, and / or

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 a substitution, in particular a single substitution, whose nucleic sequence exhibits at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, 98% and 99% identity after significant alignment with the sequence reference nuclei. These are preferably sequences whose complementary sequences are capable of hybridizing specifically with the sequences SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13 of the invention. Preferably, the specific hybridization conditions or high stringency will be such that they provide at least 70%, preferably at least 75%, 80%, ~ 85%, 90%, 95%, 98% and 99%. % identity after significant alignment between one of the two sequences and the complementary sequence of the other. Hybridization under high stringency conditions means that the temperature and ionic strength conditions are chosen such that they allow the maintenance of hybridization between two complementary DNA fragments. By way of illustration, conditions of high stringency of the hybridization step in order to define the polynucleotide fragments described above are advantageously as follows: DNA-DNA or DNA-RNA hybridization is carried out in two stages (1) prehybridization at 42 ° C. for 3 hours in phosphate buffer (20 mM, pH 7.5) containing 5 × SSC (1 × SSC corresponds to a 0.15 M NaCl solution + 0.015 M sodium citrate), 50% formamide, 7% sodium dodecyl sulfate (SDS), 10 x Denhardt's, 5% dextran sulfate and 1% salmon sperm DNA; (2) actual hybridization for 20 hours at a probe size dependent temperature (i.e., 42 C, for a probe of size>

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 100 nucleotides) followed by 2 washes for 20 minutes at 20 ° C. in 2 × SSC + 2% SDS, 1 wash for 20 minutes at 20 ° C. in 0.1 × SSC + 0.1% SDS. The last washing is carried out in 0.1 x SSC + 0.1% SDS for 30 minutes at 60 ° C. for a probe of size> 100 nucleotides. The high stringency hybridization conditions described above for a polynucleotide of defined size may be adapted by those skilled in the art for oligonucleotides of larger or smaller size, according to the teaching of Sambrook et al., 1989 .

 Among the nucleic sequences having an identity percentage of at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, 98% and 99% after significant alignment with the sequence according to according to the invention, the variant nucleic acid sequences of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, or their fragments, that is to say the set of nucleic sequences corresponding to allelic variants, that is to say individual variations of the sequences SEQ ID N 1, SEQ ID N 3, SEQ ID N 5, SEQ ID NO 7, SEQ ID NO 9, SEQ ID NO 11, SEQ ID NO 13.

 More particularly, the invention relates to a purified or isolated nucleic acid according to the present invention, characterized in that it comprises or consists of one of the sequences SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ. ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 of their complementary sequences or sequences of the RNA corresponding to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 , SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13.

The primers or probes, characterized in that they comprise a sequence of a nucleic acid according to

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 the invention are also part of the invention.

Thus, the primers or probes according to the invention are useful for detecting, identifying, assaying or amplifying nucleic acid sequence. In particular, they can make it possible to detect or discriminate the variant nucleic sequences, or to identify the genomic sequence of novel eukaryotic or prokaryotic genes, in particular bacterial genes, and more specifically of Lactobacillus bacteria, coding for an N-deoxyribosyltransferase, in using in particular an amplification method such as the PCR method, or a related method. According to the invention, the polynucleotides which can be used as probes or as primer in methods for detecting, identifying, assaying or amplifying nucleic sequences, have a minimum size of 10 bases, preferably at least 15.18, 20.25, 30.40, 50 bases. According to one embodiment, the primers according to the invention are chosen from the sequences SEQ ID No. 15 and SEQ ID No. 16.

 The polynucleotides according to the invention can thus be used as a primer and / or probe in processes using in particular the PCR (polymerase chain reaction amplification) technique (Rolfs et al., 1991). This technique requires the choice of oligonucleotide primer pairs flanking the fragment that needs to be amplified. For example, reference can be made to the technique described in US Pat. No. 4,683,202. The amplified fragments can be identified, for example after agarose or polyacrylamide gel electrophoresis, or after a chromatographic technique such as gel filtration or the

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 ion exchange chromatography, then sequences. The specificity of the amplification can be controlled by using, as primers, the nucleotide sequences of the polynucleotides of the invention and as templates, plasmids containing these sequences or the derived amplification products. The amplified nucleotide fragments can be used as reagents in hybridization reactions in order to demonstrate the presence, in a biological sample, of a target nucleic acid of sequence complementary to that of said amplified nucleotide fragments. The invention also relates to the nucleic acids that can be obtained by amplification using primers according to the invention.

 Other amplification techniques for the target nucleic acid can advantageously be used as an alternative to PCR (PCR-like) using a pair of nucleotide sequence primers according to the invention.

By PCR-like is meant all methods using direct or indirect reproductions of sequences? nucleic acids, or in which the labeling systems have been amplified, these techniques are of course known. In general it is the amplification of the DNA by a polymerase; when the original sample is an RNA, it is first necessary to perform a reverse transcription. There are currently many methods for this amplification, such as for example the SDA technique (Strand Displacement Amplification) or strand displacement amplification technique (Walker et al., 1992), the TAS (Transcription-based Amplification System) technique. described

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 by Kwoh et al. (1989), the 3SR technique (Self-Sustained Sequence Replication) described by Guatelli et al.

(1990), NASBA (Nucleic Acid Sequence Based Amplification) technique described by Kievitis et al. (1991), the TMA (Transcription Mediated Amplification) technique, the LCR (Ligase Chain Reaction) technique described by Landegren et al. (1988), the Repair Chain Reaction (RCR) technique described by Segev (1992), the CPR (Cycling Probe Reaction) technique described by Duck et al.

(1990), the Q-p-replicase amplification technique described by Miele et al. (1983). Some of these techniques have since been perfected.

 In the case where the target polynucleotide to be detected is an mRNA, it is advantageous to use, prior to carrying out an amplification reaction with the aid of the primers according to the invention or the implementation of a method detection using the probes of the invention, a reverse transcriptase enzyme to obtain a cDNA from the mRNA contained in the biological sample. The cDNA obtained will then serve as a target for the primers or probes used in the amplification or detection method according to the invention.

 The probe hybridization technique can be performed in a variety of ways (Matthews et al., 1988).

The most general method consists in immobilizing the nucleic acid extracted from the cells of different tissues or cells in culture on a support (such as nitrocellulose, nylon, polystyrene) to produce, for example, DNA chips, then to incubate in well-defined conditions, target nucleic acid immobilized with the probe. After hybridization, excess probe is removed and hybrid molecules

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 formed are detected by the appropriate method (measurement of radioactivity, fluorescence or enzymatic activity related to the probe).

 According to another mode of implementation of the nucleic probes according to the invention, the latter can be used as capture probes. In this case, a probe, called a capture probe, is immobilized on a support and serves to capture by specific hybridization the target nucleic acid obtained from the biological sample to be tested and the target nucleic acid is then detected by means of a second probe, called a detection probe, marked by an easily detectable element.

 Among the nucleic acid fragments of interest, the antisense oligonucleotides, that is to say whose structure ensures, by hybridization with the target sequence, an inhibition of the expression of the product, should be mentioned in particular. corresponding. It is also necessary to mention the sense oligonucleotides which, by interaction with proteins involved in the regulation of the expression of the corresponding product, will induce either an inhibition or an activation of this expression. The oligonucleotides according to the invention have a minimum size of 9 bases, preferably at least 10, 12, 15, 17, 20, 25, 30, 40, 50 bases.

 The probes, primers and oligonucleotides according to the invention may be labeled directly or indirectly with a radioactive or non-radioactive compound, by methods well known to those skilled in the art, in order to obtain a detectable and / or quantifiable signal. The polynucleotide sequences according to

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 The unmarked invention can be used directly as a probe or primer.

 The sequences are generally labeled to obtain sequences that can be used for many applications. The labeling of the primers or probes according to the invention is carried out by radioactive elements or by non-radioactive molecules. Among the radioactive isotopes used, mention may be made of 32P, 33P, 35S, 3H or 125I. The non-radioactive entities are selected from ligands such as biotin, avidin, streptavidin, dioxygenine, haptens, dyes, luminescent agents such as radioluminescent, chemiluminescent, bioluminescent, fluorescent, phosphorescent agents.

 The invention also comprises a method for detecting and / or assaying a polynucleotide according to the invention, in a biological sample, characterized in that it comprises the following steps: (i) isolation of the DNA from from the biological sample to be analyzed, or obtaining a cDNA from the RNA of the biological sample; (ii) specific amplification of the DNA encoding the polypeptide according to the invention using primers; (iii) analysis of the amplification products. It is also an object of the invention to provide a kit for the detection and / or assay of a nucleic acid according to the invention, in a biological sample, characterized in that it comprises the following elements: ) a pair of nucleic primers according to the invention, (ii) the reagents necessary for carrying out a DNA amplification reaction, and optionally (iii) a

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 component for verifying the sequence of the amplified fragment, more particularly a probe according to the invention.

 The invention also comprises a method for detecting and / or assaying nucleic acid according to the invention, in a biological sample, characterized in that it comprises the following steps: (i) contacting a polynucleotide according to the invention with a biological sample; (ii) detecting and / or assaying the hybrid formed between said polynucleotide and the nucleic acid of the biological sample. It is also an object of the invention to provide a kit for the detection and / or assay of nucleic acid according to the invention, in a biological sample, characterized in that it comprises the following elements: (i) a probe according to the invention, (ii) the reagents necessary for the implementation of a hybridization reaction, and / or where appropriate, (iii) a pair of primers according to the invention, as well as the reagents necessary for an amplification reaction of the DNA.

 Preferably, the biological sample according to the invention in which the detection and the assay are carried out consists of a culture medium, a ground material, a body fluid, for example a human or animal serum, blood, milk.

 The present invention also relates to recombinant cloning and / or expression vectors comprising a polynucleotide according to the invention and / or expressing a polypeptide according to the invention. Such a host cell is also an object of the invention.

 Preferably, the recombinant vectors) according to the invention are:

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the vector called pLH2 comprising the polynucleotide
SEQ ID No. 1 as present in the bacterial strain deposited at the CNCM on May 30, 2001 under No. I-2676; plasmid pLH2 contains a 1.4 kb Alu I fragment containing the gene coding for Lactobacillus helveticus CNRZ32 type II N-deoxyribosyltransferase cloned into the SmaI site of plasmid pBAM3; plasmid pLH2, which expresses this enzyme, is propagated in the auxotrophic strain PAK6 for guanine; the vector called pLH4 comprising the polynucleotide
SEQ ID No. 3 as present in the bacterial strain deposited at the CNCM on May 30, 2001 under No. I-2677; plasmid pLH4 contains a 1.6 kb AluI fragment containing the gene coding for Lactobacillus helveticus N-deoxyribosyltransferase type I CNRZ32, cloned into the SmaI site of plasmid pBAM3. Plasmid pLH4, which expresses this enzyme, is propagated in the auxotrophic strain PAK6 for guanine; the vector called pLF6 comprising the polynucleotide
SEQ ID No. 5 as present in the bacterial strain deposited at the CNCM on May 30, 2001 under No. I-2678; plasmid pLF6 contains a 1.36 kb AluI fragment containing the gene coding for Lactobacillus fermentum type II N-deoxyribosyltransferase CIP102780T. Plasmid pLF6, which expresses this enzyme is propagated in the auxotrophic strain PAK6 for guanine; the vector called pLA comprising the polynucleotide
SEQ ID No. 11 as present in the bacterial strain deposited at the CNCM on June 21, 2001 under the

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N 1-2689; the plasmid pLA corresponds to the plasmid pSU19 is cloned at the sites PstI and BamHI an insert containing the gene encoding N-deoxyribosyltransferase type II of Lactobacillus acidophilus CNRZ 1296. The plasmid is propagated in the Escherichia coli strain TG-I.

 The vectors according to the invention comprise the elements necessary for the expression, and in particular, preferably a promoter, signals for initiation and termination of the translation, as well as appropriate regions for regulating transcription. They must be able to be stably maintained in the cell and may possibly have particular signals specifying the secretion of the translated protein.

 The different control signals are chosen according to the cellular host used. For this purpose, the nucleic acid sequences according to the invention may be inserted into autonomously replicating vectors within the chosen host, or integrative vectors of the chosen host. Among the autonomously replicating systems, plasmid, cosmid, phagemid or minichromosome type systems, or viral type systems, are preferably used depending on the host cell, the viral vectors possibly being adenoviruses (Perricaudet). et al., 1992), retroviruses, lentiviruses, poxviruses or herpesviruses (Epstein et al., 1992). The person skilled in the art knows the technologies that can be used for each of these systems.

When it is desired to integrate the sequence into the chromosomes of the host cell, it is possible to use

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 example of plasmid or viral type systems; such viruses are, for example, retroviruses (Temin, 1986), or AAVs (Carter, 1993).

 Among the non-viral vectors, naked polynucleotides such as naked DNA or naked RNA according to the technique developed by the company VICAL, the bacterial artificial chromosome (BAC), the artificial chromosomes of the bacterium, are preferred. yeast (YAC, "yeast artificial chromosome") for expression in yeast, artificial chromosome (MAC) chromosomes for expression in murine cells and, preferably, artificial chromosomes of humans (HAC, "human artificial chromosome") for expression in human cells.

 Such vectors are prepared according to methods commonly used by those skilled in the art, and the resulting clones can be introduced into a suitable host by standard methods, such as, for example, lipofection, electroporation, heat shock, transformation after chemical permeabilization of the membrane, cell fusion.

 The invention furthermore comprises the host cells, in particular the eukaryotic and prokaryotic cells, transformed by the vectors according to the invention. Among the cells that can be used for the purposes of the present invention, mention may be made of bacteria and yeasts.

According to a preferred embodiment of the invention, the bacterium is chosen from the group consisting of Lactobacillus fermentum, Lactobacillus acidophilus, Lactobacillus amylovorus, Lactobacillus crispatus, Lactobacillus helveticum, Lactobacillus lactis,

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 Escherichia coli, Bacillus subtilis, Campylobacter pylori, Helicobacter pylori, Agrobacterium tumefaciens, Staphylococcus aureus, Thermophilus aquaticus, Azorhizobium caulinodans, Rhizobium leguminosarum, Neisseria gonorrhoeae, Neisseria meningitis. According to a preferred embodiment of the invention, the bacterium is Lactobacillus. According to a preferred embodiment, it is a question of: the bacterium transformed with the plasmid pLH2 comprising the polynucleotide SEQ ID No. 1 as deposited at the CNCM on May 30, 2001 under No. I-2676; the bacterium transformed with plasmid pLH4 comprising the polynucleotide SEQ ID No. 3 as deposited at the CNCM on May 30, 2001 under No. I-2677; the bacterium transformed with the plasmid pLF6 comprising the polynucleotide SEQ ID No. 5 as deposited at the CNCM on May 30, 2001 under No. I-2678; the bacterium transformed with the plasmid pLA comprising the polynucleotide SEQ ID No. 11 as deposited at the CNCM on June 21 under No. I-2689.

 According to another preferred embodiment the bacterium is Escherichia coli. According to another embodiment of the invention, the cell is a yeast which is preferably Saccharomyces cerevisiae, Saccharomyces pombe, Candida albicans.

 Among the host cells according to the invention, it is also worth mentioning insect cells, animal or plant cells.

 Preferably, the cell according to the invention is devoid of enzymatic activity capable of degrading said deoxyribonucleoside precursor or said deoxyribonucleoside obtained by bioenzymatic reaction

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 catalyzed by a polypeptide according to the invention.

Alternatively, said host cell may be provided with additional bio-enzymatic activities intended to convert the deoxyribonucleoside precursor, and / or the deoxyribonucleoside obtained by the bioenzymatic reaction catalyzed by the polypeptide according to the invention. Among these additional bio-enzymatic activities, it is necessary to mention phosphorylation, sulfation, acetylation, succinylation, methylation.

 The nucleic acid sequence encoding the N deoxyribosyltransferases according to the invention is either naturally present in said cell or is introduced into said cell by recombinant DNA techniques known to those skilled in the art. According to a preferred embodiment, the nucleic acid sequence introduced into said cell by the recombinant DNA techniques and which codes for an N deoxyribosyltransferase according to the invention is heterologous. Heterologous nucleic acid sequence is understood to mean; a nucleic acid sequence which is not naturally present in the cell according to the invention.

 The present invention also relates to metazoan organisms, plant or animal, preferably mammals, except humans, comprising one of said transformed cells according to the invention.

Among the animals according to the invention, rodents, in particular mice, rats or rabbits, expressing at least one polypeptide according to the invention are preferred.

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 The preferably bacterial cells, or fungal yeast in particular, and the metazoan organisms according to the invention are usable in a method for producing N-deoxyribosyltransferase according to the invention. The method for producing a polypeptide of the invention in recombinant form, itself included in the present invention, is characterized in that the transformed cells, in particular the cells of the present invention, are cultivated under conditions allowing the expression and optionally the secretion of a recombinant polypeptide encoded by a nucleic acid sequence according to the invention, and that said recombinant polypeptide is recovered. The recombinant polypeptides obtainable by this production method are also part of the invention. They may be in glycosylated or non-glycosylated form and may or may not have the tertiary structure of the natural protein. The sequences of the recombinant polypeptides may also be modified in order to improve their solubility, in particular in aqueous solvents. Such modifications are known to those skilled in the art, for example the deletion of hydrophobic domains or the substitution of amino acids. hydrophobic by hydrophilic amino acids.

 These polypeptides can be produced from the nucleic acid sequences defined above, according to recombinant polypeptide production techniques known to those skilled in the art. In this case, the nucleic acid sequence used is placed under

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 the control of signals allowing its expression in a cellular host.

 An efficient system for producing a recombinant polypeptide requires having a vector and a host cell according to the invention. These cells can be obtained by introducing into host cells a nucleotide sequence inserted into a vector as defined above, and then culturing said cells under conditions allowing the replication and / or expression of the transfected nucleotide sequence.

 The methods used for the purification of a recombinant polypeptide are known to those skilled in the art. The recombinant polypeptide can be purified from lysates and cell extracts, from the supernatant of the culture medium, by methods used individually or in combination, such as fractionation, chromatography methods, immunoaffinity techniques using monoclonal or polyclonal antibodies, etc. A preferred variant is to produce a recombinant polypeptide fused to a carrier protein (chimeric protein). The advantage of this system is that it allows for stabilization and decrease of proteolysis of the recombinant product, increase in solubility during in vitro renaturation and / or simplification of purification when the fusion partner has a affinity for a specific ligand.

 The polypeptides according to the present invention can also be obtained by chemical synthesis using one of the many peptide syntheses

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 known, for example techniques using solid phases or techniques using partial solid phases, by condensation of fragments or by synthesis in conventional solution. The polypeptides obtained by chemical synthesis and which may comprise corresponding non-natural amino acids are also included in the invention.

 The polypeptides according to the invention make it possible to prepare monoclonal or polyclonal antibodies. It is therefore also an object of the present invention to provide a monoclonal or polyclonal antibody and its fragments, characterized in that they bind selectively and / or specifically a polypeptide according to the invention.

Chimeric antibodies, humanized antibodies and single chain antibodies are also part of the invention. The antibody fragments according to the invention are preferably Fab, F (ab ') 2, Fc or Fv fragments.

The polyclonal antibodies may be prepared, for example by immunization of an animal, in particular a mouse, with a polypeptide according to the invention combined with an adjuvant of the immune response, and then purification of the specific antibodies contained in the serum of the immunized animals. on an affinity column on which has previously been fixed the polypeptide having served as antigen. The polyclonal antibodies according to the invention may also be prepared by purification on an affinity column, on which a polypeptide according to the invention has previously been immobilized. The monoclonal antibodies can advantageously be prepared from hybridomas according to the technique described by Kohler and Milstein in 1975.

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 According to a particular embodiment of the invention, the antibody is capable of inhibiting the interaction between the N-deoxyribosyltransferase of the invention and its substrate in order to alter the physiological function of said N-deoxyribosyl transferase polypeptide.

 The invention also relates to methods for detecting and / or purifying a polypeptide according to the invention, characterized in that they use an antibody according to the invention. The invention further comprises purified polypeptides, characterized in that they are obtained by a method according to the invention.

 Moreover, in addition to their use for the purification of polypeptides, the antibodies of the invention, in particular the monoclonal antibodies, can also be used for the detection of these polypeptides in a biological sample.

 For these different uses, the antibodies of the invention may also be labeled in the same manner as described above for the nucleic acid probes of the invention and, preferably, with enzymatic, fluorescent or radioactive type labeling.

 The antibodies of the invention also constitute a means for analyzing the expression of polypeptide according to the invention, for example by immunofluorescence, gold labeling, enzymatic immunoconjugates. More generally, the antibodies of the invention may be advantageously used in any situation where the expression of a polypeptide according to the invention, normal or mutated, must be observed, and more particularly in immunocytochemistry, in immunohistochemistry or in of the

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 western blotting experiments. Thus, a method for detecting a polypeptide according to the invention in a biological sample, comprising the steps of bringing the biological sample into contact with an antibody according to the invention and of demonstrating the antigen-antibody complex formed is also an object of the invention.

 It is also an object of the present invention to provide a process for enzymatic synthesis in vitro or in vivo of deoxyribonucleotides, characterized in that it comprises at least one reaction step catalyzed by at least one N-deoxyribosyltransferase according to the invention. The method according to the invention is characterized in that said N-deoxyribosyl transferase catalyzes the exchange of a first nucleobase present in a deoxyribonucleoside by a second nucleobase.

 According to a preferred embodiment of the invention, said second nucleobase is selected from the group consisting of purines linked by N9, pyrimidines linked by N1, azines linked by NI, imidazoles linked by NI, said second --- nucleobases capable of carrying substitutions of the hydrogens at unbound positions. Preferably, said second nucleobase is selected from the group consisting of 6-methyl purine, 2-amino-6-methylmercaptopurine, 6-dimethylaminopurine, 5-azacytidine, 2,6-dichloropurine, 6-chloroguanine, 6-chloropurine, 6-aza -thymine, 5-fluorouracil, ethyl-4-amino-5-imidazole carboxylate, imidazole-4-carboxamide and 1,2,4-triazole-3-carboxamide.

The said first nucleobase is, for its part, preferably selected from the group consisting of

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 adenine, guanine, thymine, uracil and hypoxanthine. These lists are not exhaustive, and it is evident that natural or non-natural analogues of nucleobases may be employed in the present invention as a substrate of an N-deoxyribosyl transferase of the invention.

 Optionally, the in vivo method according to the invention is characterized in that it further comprises the step of introducing into the host cell the first nucleobase present in a deoxyribonucleoside.

 Optionally, the in vivo method according to the invention is characterized in that it further comprises the step of introducing into the host cell the second nucleobase present in a deoxyribonucleoside.

 Optionally, the in vivo method according to the invention is characterized in that it further comprises the step of introducing into the host cell the first nucleobase present in a deoxyribonucleoside and the second nucleobase simultaneously and / or shifted in the time.

 The deoxyribonucleosides that can be produced in large quantities and inexpensively by the biosynthetic method according to the invention therefore constitute compounds of interest intended for the preventive or curative treatment of human or animal, tumor or viral pathologies such as AIDS. (acquired human immunodeficiency syndrome), bacterial, parasitic or fungal.

Alternatively, these deoxyribonucleosides, which can be produced in large quantities and inexpensively by the biosynthetic method according to the invention, constitute herbicides and insecticides.

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 Other features and advantages of the invention appear in the following description with the examples shown below.

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EXAMPLES
1. MATERIAL AND METHODS
1. 1. Strains and culture conditions:
The lactic acid bacteria strains used come from the CNRZ collection (National Center for Zootechnical Research), Dairy Research and Applied Genetics Unit, INRA, Jouy en Josas. They are cultured in MRS broth (de Man et al., J. Appl., Bacteriol., 23: 130-135, 1960) and incubated at 30 ° C., 37 ° C. or 42 ° C. depending on the species. The strain of Escherichia coli TG1, provided by Stratagene, is cultured in LB (Luria broth base 10 g / l, Agar-agar 16 g / l) with stirring and at 37 ° C.

1. 2. Preparation of the total cellular DNA of lactic acid bacteria:
The cultures at the end of the exponential phase are centrifuged for 5 minutes at 13000 g. The pellet corresponding to a culture of 2 ml is taken up in 200 l of TES (50 mM Tris, pH8, 10 mM EDTA, pH8, 250 mM sucrose) containing 20 g / ml of lysozyme and 50 U / ml of mutanolysin (Sigma ). After an incubation of one hour at 37 ° C., the clarification of the preparation is obtained by adding 60 μl of 20% SDS.

 The extraction of the nucleic acids is carried out by adding to the lysate 500 l of phenol saturated with water, pH 8, added with hydroxyquinoline 0.1% and 100 l of a mixture of chloroform-isoamyl alcohol (24/1, V / V ).

The solution is homogenized and then centrifuged for 10 minutes at 13000 g and at room temperature. The sentence

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 superior, limpid and containing the nucleic acids is kept. On the latter, the extraction is repeated three times in order to eliminate unwanted cellular constituents. The traces of phenol are eliminated by adding 500 μl of chloroform-isoamyl alcohol to the aqueous phase. After homogenization and centrifugation for 3 minutes at 15,000 g and at 4 ° C., the nucleic acids contained in the upper aqueous phase are precipitated by adding a volume of cold isopropanol. After incubation for one hour at -20 ° C., centrifugation is carried out for 20 minutes at 15,000 g and at 4 ° C. The isopropanol is removed and replaced with 500 l of 70% ethanol. A final centrifugation of 10 minutes at 15000 g and at 4 C makes it possible to recover a nucleic acid pellet. This is allowed to dry in an evaporator and resuspended in 200 l of sterile water containing 10 l of 10 g / l RNase. After incubation for 15 minutes at 37 ° C. for the action of the RNA-degrading enzyme, 10 μl of the DNA solution are electrophoretically migrated in a 0.8% agarose gel in order to evaluate their concentration and the quality.

1.3. DNA polymerase chain reaction (PCR):
The polymerase chain reactions (PCR) are carried out in a reaction volume of 100 μl containing 20 to 100 ng of DNA, 0.5 M of primers, 200 μM of dNTPs (dATP, dCTP, dGTP, dTTP), in 10 mM TrisHC1 pH 9 buffer, 50 mM KCl, 1.5 mM MgCl 2, 0.002% BSA and 2.5 units of Taq polymerase. Thirty cycles of amplification were applied (Gene Amp PCR

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systems 2400, Perkin Elmer). The inventors have defined two primers ntd 1 (SEQ ID No. 15) and ntd2 (SEQ ID No. 16) from the ntd sequence of Lactobacillus leichmanii described by Huck (personal communication):

<Tb>
<tb> ntd <SEP> 1 <SEP>5'-AGA<SEP> CGA <SEP> TCT <SEP> ACT <SEP> TCG <SEP> GTG-3 '<SEP> 18 <SEP> bases <SEP> Tm = <SEP> 54 C
<tb> ntd <SEP> 2 <SEP>5'-ACG<SEP> GCA <SEP> CCT <SEP> TCG <SEP> TAG <SEP> AAG-3 '<SEP> 18 <SEP> bases <SEP> Tm = <SEP> 56 C
<Tb>

1. 4. Southern Hybridization:
Enzymatic restriction of DNAs. The total DNAs are digested with one or more restriction enzymes. The enzymes used are: BamHI, BglII, ClaI, EcoRI, HindIII, HpaI, NcoI, NotI, PstI, XbaI, XhoI (Bio-Lab). The digestion is carried out in a final volume of 40 l containing 70 U of enzyme, 4 μl of NEB buffer (Bio-Lab) 10 × and 4 to 8 g of DNA. The incubation is carried out for 1h30 to 37 C.

 Transfer of the DNA on a membrane. The total DNA fragments from enzymatic digestion are separated using a 0.7% agarose gel. After migration, the agarose gel is stirred in a depuration solution (0.25N HCl) for 30 minutes. This method thus allows the transfer of DNA fragments greater than 10 kbs. After rinsing with water, the DNA is denatured by placing the gel for 40 minutes in a solution of 5M NaCl, 0.5M NaOH. The gel is rinsed with water and then incubated for another 30 minutes in a neutralization solution, 1.5M NaCl, 0.5M Tris HCl; pH 7.5. The DNAs are transferred by capillarity onto a positively charged nylon membrane (Hybond N +, Amersham). They are eluted by an upward flow of

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 SSC 20X (0.3M trisodium citrate, 3M NaCl, pH7). After transfer, the DNAs are covalently attached to the membrane using a Stratalinker 2400 UV device (Stratagene).

 Preparation of the probe. The probe used is an internal fragment of the ntd gene of Lactobacillus helveticus amplified by PCR. The probe is purified using the Wizard kit (Promega) to eliminate PCR primers. The necessary probe concentration is 10 ng / l. The labeling of the DNA is carried out using the ECL labeling kit (Amersham). To do this, the DNA is denatured by heating for 5 minutes at 100 ° C. and immediately cooled in ice. One volume of labeling reagent (peroxidase) and then one volume of glutaraldehyde solution are added. This solution is incubated for 10 minutes at 37 ° C to covalently attach the peroxidase to the DNA.

 Hybridization and revelation. After prehybridization for one hour at 42 ° C. in hybridization buffer, the membrane is hybridized for 16 hours at 42 ° C. in the presence of the labeled probe. In order to eliminate the nonspecifically fixed probe, the membrane is washed for 20 minutes at 42 ° C. in two successive baths of buffer: urea 6M - SDS 0.4% - SSC 0.5X, then rinsed for 5 minutes in two successive baths of buffer: sodium 0.3M - NaCl 3M pH 7. The revelation is carried out by autoradiography according to the protocol of the kit ECL. A first developing reagent containing hydrogen peroxide is reduced by the peroxidase attached to the probe. Then the luminol contained in a second reagent

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 The revelation is then oxidized, producing light that impresses the autoradiographic film.

1. 5. Cloning of PCR fragments:
The ntd homologous genes amplified by PCR are inserted into the plasmid vector pBluescript II SK + E. coli TG1 (Stratagene). This plasmid is previously restricted to its unique site by the enzyme EcoRV (GibcoBRL) which creates blunt ends. The digestion mixture is carried out in a volume of 30 l, containing 4 l of DNA. The amplified DNA fragments to be cloned should have their 5 'and 3' ends blunt to allow cloning. The preparation of the blunt ends of 50 l of purified PCR products using the Wizard kit (Promega) is carried out in a reaction volume of 100 l containing 3.6 units of phage T4 DNA polymerase (Bio-Lab) and 6 DNA polymerase I (or Klenow fragment) units (Bio-Lab), having no 5 '>3' exonuclease activity. The polymerization is carried out for 20 minutes at 11 ° C., then the enzymes are inactivated after 10 minutes at 75 ° C. The DNA is then precipitated in the presence of two volumes of 100% ethanol, glycogen and 10% of sodium acetate. 3M, pH 4.8. The mixture is placed for one hour at -20 ° C. and then centrifuged for 20 minutes at 15,000 g. The pellet is rinsed with 250 l of 70% ethanol, centrifuged again for 10 minutes at 15000 g, dried in an evaporator and resuspended in 20 l of sterile water.

 The restricted DNA of the plasmid pBS-SK + and the amplified fragment are co-migrated on an agarose gel to

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 0.7% in order to evaluate their respective concentrations: the number of molecules of the fragment to be cloned must be three to four times greater than that of the plasmid. The ligation is in a volume of 10 l containing 60 ng of insert, 26 ng of plasmid pBS-SK + restricted, 2 units of ligase (T4 DNA ligase, Boehringer-Mannheim), overnight at 16 C. The products of ligation are dialyzed on a 0.025 m Milipore filter so as to remove salts and thus avoid arcing during electroporation.

1. 6. Transformation:
Preparation of electro-competent cells of E. coli strain TG1 coli. From 5 ml of a night culture at 37 C with stirring, 500 ml of LB medium are inoculated. The culture is stirred at 37 C until an OD 600 nm = 1 is reached. It is then cooled for 2 hours in ice and then centrifuged for 10 minutes at 5000 rpm. The supernatant is removed, the pellet is taken up in 400 ml of cold water. This preparation is centrifuged for 10 minutes at cold and 5000 rpm. The pellet obtained is again taken up in 250 ml of cold water. After a centrifugation of 10 minutes, the pellet is taken up in 25 ml of cold water and the cells are resuspended in a final volume of 1 ml of glycerol 10%, and divided into an aliquot before being frozen quickly in liquid nitrogen.

 Transformation by electroporation and selection of clones. Electro-competent cells stored at -80 C are thawed in ice and then

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 contact with 5 l of ligation mixture in an electroporation cuvette. The Gene-Pulser (Bio-Rad) is set at 200 volts, 25 mF, 250 ohms. The cells are then electroporated. 1 ml of a solution of SOC (bactopeptone 20 g / L, yeast extract 5 g / L, 5 mM NaCl 2 / L, KCl 1M 2 .5 ml / L, MgCl 2, 10 ml / l, 1M MgSO 4 10 ml / L) are added, containing 0.4% glucose in the cell suspension which is incubated at 37 ° C. for one hour. The cells are then plated on a selective medium LB-Xgal (5-bromo-4-chloro-3-indolyls-D-galatoside at 1 g / ml) -IPTG (isopropythio-β-D-galactoside, 1 g / ml) Amicillin (50 g / ml), and incubated at 37 ° C overnight.

 Rapid extraction of plasmid DNA from recombinant E. coli clones by alkaline lysis. The E. coli cells transformed and cultured in LB medium containing ampicillin (100 g / ml) are harvested by centrifugation at 15000 g for 10 minutes at 4 ° C. They are resuspended in 50 ml of a 50 mM sucrose solution. 25 mM Tris-HCl, pH 8, 10 mM EDTA, pH 8. The alkaline lysis and denaturation of the DNA is carried out by adding 200 l of 0. 2N NaOH, 1% SDS. The whole is left for 1 minute at room temperature after adding 200 l of chloroform. Then, 150 l of 5M potassium acetate solution, glacial acetic acid are added.

The whole is centrifuged for 15 min at 13000 g at 4 C. The aqueous phase containing the DNA is precipitated in the presence of 2 volumes of 100% ethanol and then centrifuged for 20 minutes at 13000 g at 4 C. The pellet is washed with 70% ethanol, centrifuged for 10 minutes at 13000 g then

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 resuspended in 30 μl / ml RNase-containing sterile water thread.

1. 7. Inverse PCR:
Inverse PCR amplifies the regions flanking a DNA fragment of known sequence. This technique takes place in three stages:
Digestion of the DNA template. The DNA template is digested with one or two restriction enzymes selected so that they do not cut into the known gene sequence and provide a fragment of suitable size (1-3 kb). In order to choose a suitable enzyme, the total DNA is digested separately by several enzymes. Southern hybridizations are then carried out using the known sequence DNA fragment as a probe. Digests for which the hybrid probe with a fragment size of 1 to 3 kb, are used for the reverse PCR. The DNA fragments obtained by digestion are circularized.

For this, 100 units of T4 DNA ligase and 100 μl of ligation buffer are added to 4 to 8 g of DNA in a final volume of 1 ml. The ligation mixture is incubated at 15 ° C overnight. The ligated DNA is then precipitated with 100 l of 3M sodium acetate, pH 4.8, 700 l of isopropanol and 2 l of glycogen then centrifuged for 30 minutes at 13000 g, at 4 ° C. The pellet is rinsed in 300 μl. 70% ethanol and centrifuged for 10 minutes at 13000 g at 4 C. The pellet is taken up in 25 l of ultrapure water.

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 Amplification of circularized DNAs using divergent primers. The polymerase chain reactions are carried out in a reaction volume of 100 (containing 20 to 100 ng DNA, 0.5 M primers, 200 M dNTPs (dATP, dCTP, dGTP, dTTP), in a buffer Tris-HCl pH 9 at 10 mM, KCl at 50 mM, MgCl2 at 1.5 mM BSA at 0.002 and 2.5 units of the Taq polymerase.

The amplification is performed under the following conditions:
94 C 3 minutes
94 C 30 seconds
50 to 60 C (depending on the Tm of the primers used) 1 minute 25 cycles
72 C 3 minutes (Gene Amp PCR systems 2400, Perkin Elmer).

 Sequencing. The PCR fragments are purified by the Wizard kit (Promega) to remove unincorporated oligonucleotides, salts and Taq polymerase. Sequencing is performed using a 373A automatic sequencer (Applied Biosystems-Perkin Elmer) using an ABI PRISM Dye Terminator kit (Perkin Elmer), based on the incorporation of fluorescent phosphate deoxynucleotides during the elongation phase. primers. Sequence reactions are performed in a 20 l reaction volume containing 30 ng of DNA, 4 l of DyeT Mix (Perkin Elmer Biosystems) and 0.1 ml of primer.

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Cycle: 96 C 1 minute 96 C 10 seconds 50 C 5 seconds 25 cycles 60 C 4 minutes
3M sodium acetate, pH 4.6, 50L of 95% ethanol and 1L of glycogen are added to each sequence reaction, the solution is left for 15 minutes at room temperature and then centrifuged for 20 minutes. at 13000 g The pellet is then rinsed with 250 μl of 70% ethanol and then centrifuged for 10 minutes at 13000 g The pellet is then taken up in 6 l of sequence blue (83% formamide, 8.3 mM EDTA, dextran blue 2000000 (Sigma) 0.5%) The samples are denatured for 3 minutes at 90 ° C. and 3 l are deposited on 4% acrylamide gel.

 2. A UNIQUE NUTRITIONAL SCREEN FOR THE TWO CLASSES OF N-DESOXYRIBOSYLTRANSFERASE IN ESCHERICHIA COLI.

 A functional screen for selecting guanine production was established in E. coli, deleting the two genes of the guaBA operon that controls the conversion of IMP to XMP and GMP, as well as those of the deoCABD operon that controls the production of guanine. degradation of the deoxynucleosides, to give the strain PAK 6. The genome of E. coli specifies an activity to convert the G base to GMP (guanine phosphoribosyltransferase encoded by the gpt gene), as well as an activity to release the G base from

<Desc / Clms Page number 43>

deoxynucleoside dR-G (purine nucleoside phosphorylase encoded by the deoD gene, in the deo operon). The PAK 6 strain therefore has a guanine (G) requirement which can not be satisfied by the addition of deoxyguanosine (dR-G). The use of deoxyguanosine (dR-G) may, however, be selected if an N deoxyribosyltransferase activity is expressed in the strain PAK6, to carry out the exchange: dR-G + A <-> dR-A + G
This exchange between two purine bases can be catalyzed by the two classes of enzyme. In fact, the introduction of the L. leichmannii ntd gene into the PAK 6 strain makes it possible to satisfy the guanine requirement using deoxyguanosine (dR-G) and adenine (A).

 3. FUNCTIONAL CLONING OF L. PTD GENE

 HELVETICUS.

 DNA fragments of size between 1 and 2kb obtained by partial digestion (AluI) of L. helveticus CNRZ 32 were ligated into a plasmid C-olEl (pUC type digested with SmaI and dephosphorylated) and the mixture was used to transform the strain PAK6. The transforming clones were selected in a glucose mineral medium supplemented with deoxyguanosine (dR-G) and adenine (A) at a final concentration of 0.3 mM.

 One of the transformant clones was found to propagate a plasmid containing an insert controlling a Class I Ndeoxyribosyltransferase activity and deviating from the restriction profile of the L. helveticus ntd gene. The sequence of this insert revealed a gene specifying a polypeptide of 167 amino acids with a molecular weight of 18790. 70 Daltons exhibiting a

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 similarity of 28.6% with the L. leichmanii NTD polypeptide. The sequence of this gene designated ptd deviates from that of genes ntd making their hybridization impossible (7.5% identity).

 Incidentally, the L. helveticus ntd gene controlling class II N-deoxyribosyltransferase activity was again isolated from the selected transforming clones.

 4. FUNCTIONAL CLONING OF L. NTD GENE

Fermentum.

 The same cloning and nutritional selection operations were carried out from the genomic DNA of L. fermentum strain CIP 102980T. The selected transforming clones were found to propagate a plasmid whose inserts with similar restriction profiles controlled class II N deoxyribosyltransferase activity. The sequence of one of these inserts revealed a gene specifying a polypeptide of 168 amino acids with a molecular weight of 18878. Daltons having a 32.9% similarity with the L. leichmanii NTD polypeptide and 36.7% with L. helveticus PTD polypeptide. The sequence of this gene deviates from that of the genes ntd and ptd making their hybridization impossible. The L. fermentum NTD polypeptide has a more active evolutionary relationship for the class I enzyme (L. helveticus PTD) and class II functional affiliation, suggesting early evolutionary divergence in the evolution of these enzymes. Its enzymatic activity could be

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 different from those of other species, and lend itself to the preparation of a larger spectrum of nucleosides.

 5. INVERSE PCR CLONING OF FOUR NTD GENES.

 Using degenerate oligonucleotides from regions of the amino acid sequence of the L. leichmanii NTD polypeptide (Hück, 1997) an internal fragment of the L. helveticus ntd gene was amplified. From this fragment, oligonucleotides were synthesized so as to obtain the entire gene by inverse PCR.

 From the two ntd sequences of L. leichmannii and L. helveticus, we have redefined consensus primers to isolate the ntd genes of other lactobacillus species such as L. acidophilus, L. crispatus, L. amylovorus with the same approach as the one described above.

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Table 1: Growth of the PAK 6 Strain Which Expresses Non-N-Deoxyribosyltransferase Activity on Glucose Mineral Medium (In Vivo) and Enzyme Activity of the Corresponding Crude Extracts (In Vitro)

<Tb>
<tb> in <SEP> in
<tb> vivo <SEP> vitro
<Tb>

~h'.........................-.-....................................."""".....n....""""'"'''''''''''''''''''''''''''''' .....-................. "...n.- ......., ".--- ~.-

~ H '.........................-.-................... .................."""".....not....""""'"'''''''''''''''''''''''''''''' .....-................. "...not.- .. ....., ".--- ~ .-

<Tb>
<tb> A <SEP> G <SEP> dG <SEP> dG <SEP> + <SEP> A <SEP> dC <SEP> + <SEP> T <SEP> dG <SEP> + <SEP> A <SEP > dC <SEP> + <SEP> A <SEP>
<tb> PAK <SEP> 6 <SEP> - <SEP> + <SEP> ~ <SEP> - <SEP> - <SEP> - <SEP> pSU19
<tb> PAK <SEP> 6 <SEP> ntd <SEP> - <SEP> + <SEP> + <SEP> + <SEP> + <SEP> +
<tb> Ll
<tb> PAK <SEP> 6 <SEP> ntd <SEP> - <SEP> + <SEP> + <SEP> + <SEP> + <SEP> +
<tb> Lh
<tb> PAK <SEP> 6 <SEP> ptd <SEP> - <SEP> + <SEP> ~ <SEP> ± <SEP> +
<tb> Lh
<tb> PAK <SEP> 6 <SEP> ntd <SEP> - <SEP> + <SEP> + <SEP> + <SEP> + <SEP> +
<tb> Lf
<Tb>
(+) growth (-) absence of growth 'PAK 6: MG1655 AguaBA :: Apra, Adeo ntd L1: gene encoding Lactobacillus leichmannii ntd deoxyribosyltransferase Lh: coding for Lactobacillus helveticus ntd deoxyribosyltransferase ntd Lf: coding for N-deoxyribosyltransferase of Lactobacillus fermentum ptd Lh: Gene encoding the purine deoxyribosyltransferase of Lactobacillus helveticus

<Desc / Clms Page number 47>

 A: adenine; G: guanine; T: thymine; dG: deoxyguanosine; dC: deoxycytidine

<Desc / Clms Page number 48>

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Claims (34)

An isolated or purified polypeptide of Lactobacillus having at least one N-deoxyribosyltransferase activity of amino acid sequence selected from the sequences SEQ ID N 4, SEQ ID N 6, SEQ ID N 8, SEQ ID N 10, SEQ ID N 12 .  2. Isolated polypeptide according to claim 1, characterized in that the polypeptide of SEQ ID No. 4 is encoded by the N-deoxyribosyltransferase encoded by the ptd Lh gene of Lactobacillus helveticus.  3. Isolated polypeptide according to claim 1, characterized in that the polypeptide of SEQ ID No. 6 is encoded by the N-deoxyribosyltransferase encoded by the ntd Lf gene of Lactobacillus fermentum.  4. Isolated polypeptide according to claim 1, characterized in that the polypeptide of SEQ ID No. 8 is encoded by the N-deoxyribosyltransferase encoded by the ntd gene of Lactobacillus crispatus.  5. isolated polypeptide according to claim 1 characterized in that the polypeptide of SEQ ID N 10 is encoded by N-deoxyribosyltransferase encoded by the ntd gene of Lactobacillus amylovorus.  6. Isolated polypeptide according to claim 1, characterized in that the polypeptide of SEQ ID No. 12 is encoded by the N-deoxyribosyltransferase encoded by the ntd gene of Lactobacillus acidophilus.  7. Isolated polypeptide characterized in that <Desc / Clms Page number 51>  comprises a polypeptide selected from: a) a polypeptide of sequence SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 b) an amino acid sequence polypeptide variant polypeptide defined in a); c) a polypeptide homologous to the polypeptide defined in a) or b) and having at least 80% identity with said polypeptide of a), d) a fragment of at least 15 consecutive amino acids of a polypeptide defined in a) ; e) a biologically active fragment of a polypeptide defined in a), b) or c).  8. Polypeptide according to one of claims 1 to 7 characterized in that it satisfies the requirement of guanine strain PAK6 deposited at the CNCM May 2, 2001 under the? 1-2664.  9. purified or isolated polynucleotide characterized in that it encodes a polypeptide according to one of claims 1 to 8.  10. The polynucleotide according to claim 9 of sequence SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13.  11. Isolated polynucleotide characterized in that it comprises a polynucleotide chosen from a) SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13. b) sequence of a fragment of at least 15 consecutive nucleotides of the sequence SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13; <Desc / Clms Page number 52>  c) a nucleic sequence having an identity percentage of at least 70%, after optimal alignment with a sequence defined in a) or b); d) the complementary sequence or the RNA sequence corresponding to a sequence as defined in a), b) or c).  12. Polynucleotide according to one of claims 9 to 11 characterized in that its expression in the PAK6 strains makes it possible to satisfy the guanine requirement of said strain.  13. Use of a polynucleotide according to claim 11 as a primer for the amplification or polymerization of nucleic acid sequences of N deoxyribosyltransferases.  14. Use of a polynucleotide according to one of claims 9 to 12 as a probe for the detection of nucleic acid sequences of N deoxyribosyltransferases.  A recombinant cloning and / or expression vector comprising a polynucleotide according to one of claims 9 to 12 or expressing a polypeptide according to any one of claims 1 to 8.  16. Recombinant vector called pLH4 comprising the polynucleotide SEQ ID No. 3 as present in the bacterial strain deposited at the CNCM on May 30, 2001 under No. I-2677.  17. Recombinant vector called pLF6 comprising the <Desc / Clms Page number 53>  polynucleotide SEQ ID N 5 as present in the bacterial strain deposited at the CNCM on May 30, 2001 under No. I-2678.  18. Recombinant vector called pLA comprising the polynucleotide SEQ ID No. 11 as present in the bacterial strain deposited at the CNCM on June 21, 2001 under No. I-2689.  19. Host cell, characterized in that it is transformed with a vector according to one of claims 15 to 18.  20. Bacterium transformed with the pLH4 vector comprising the polynucleotide SEQ ID No. 3 as deposited at the CNCM on May 30, 2001 under No. I-2677.  21. Bacterium transformed with the pLF6 vector comprising the polynucleotide SEQ ID No. 5 as deposited at the CNCM on May 30, 2001 under No. I-2678.  22. Bacterium transformed with the pLA vector comprising the polynucleotide SEQ ID No. 11 as deposited at the CNCM on June 21, 2001 under No. I-2689.  23. Metazoan animal or plant organism, except man, characterized in that it comprises a cell according to claim 19.  24. Process for the preparation of a recombinant polypeptide, characterized in that a host cell according to claim 19 or a bacterium according to one of claims 20 to 22 is cultured in <Desc / Clms Page number 54>  conditions allowing the expression and optionally the secretion of said recombinant polypeptide and recovering said recombinant polypeptide.  25. A recombinant polypeptide obtainable by a process according to claim 24.  26. Monoclonal or polyclonal antibody and its fragments characterized in that it selectively binds a polypeptide according to one of claims 1 to 8 or 25.  27. Process for the enzymatic synthesis in vitro or in vivo of deoxyribonucleotides, characterized in that it comprises at least one reaction step catalyzed by an N-deoxyribosyltransferase according to any one of Claims 1 to 8.  28. The method of claim 27 characterized in that said N-deoxyribosyltransferase catalyzes the exchange of a first nucleobase present in a deoxyribonucleoside by a second nucleobase.  29. The method according to claim 28, wherein said second nucleobase is selected from the group consisting of N9-linked purines, N1-linked pyrimidines, NI-linked azines, and NI-linked imidazoles, said second nucleobases being substitutions of the hydrogens at unbound positions.  30. The method of claim 29 characterized in that said second nucleobase is selected from the group consisting of 6-methyl purine, 2-amino-6- <Desc / Clms Page number 55>  methylmercaptopurine, 6-dimethylaminopurine, azacytidine, 2,6-dichloropurine, 6-chloroguanine, 6chloropurine, 6-aza-thymine, 5-fluorouracil, 4-ethylamino-5-imidazole carboxylate, imidazole-4-carboxamide, and 1,2-dimethylaminopurine. , 4-triazole-3-carboxamide.  31. The method of claim 28 characterized in that said first nucleobase is selected from the group consisting of adenine, guanine, thymine, uracil and hypoxanthine.  32. In vivo method according to one of claims 28 to 31 characterized in that it further comprises the step of introducing into the host cell the first nucleobase present in a deoxyribonucleoside.  33. In vivo method according to one of claims 28 to 31 characterized in that it further comprises the step of introducing into the host cell the second nucleobase present in a deoxyribonucleoside.  34. In vivo method according to one of claims 28 to 31 characterized in that it further comprises the step of introducing into the host cell the first nucleobase present in a deoxyribonucleoside and the second nucleobase simultaneously and / or shifted in time