AU2002362377B2 - Lactobacillus N-deoxyribosyl transferases, corresponding nucleotide sequences and their uses - Google Patents

Abstract

Isolated or purified polypeptides (I) from Lactobacilluswith at least one N-deoxyribosyltransferase activity which is any of sequences S2, S4, S8, S10, or S12 with fully defined 158, 167, 84, 133 and 159 amino acid sequences, respectively, given in the specification, are new. Independent claims are also included for the following: (1) isolated polypeptides (Ia) that are: (a) (I); (b) variants of (a); (c) homologs having at least 80% homology with (a); (d) fragments of (a) having at least 15 consecutive aa; and (e) biologically active fragments of (a)-(c); (2) pure or isolated nucleic acid (II) that encodes (I) or (Ia); (3) recombinant cloning and expression vector that contains (II) or expresses (I) or (Ia); (4) host cell transformed with a vector of (3); (5) metazoan animal or plant, excluding humans, that contain a cell of (4); (6) preparing recombinant polypeptide (Ib) by culturing cells of (4); (7) (Ib) produced by method (6); (8) mono- or poly-clonal antibodies (Ab), or their fragments, that bind selectively to (I)-(Ib); and (9) in vitroor in vivoenzymatic synthesis of deoxyribonucleotides that includes a step catalyzed by the new deoxyribosyltransferases. ACTIVITY : Antibacterial; Antiviral; Antifungal; Anti-HIV; Insecticide; Heribicide; Cytostatic. No biological data is given. MECHANISM OF ACTION : None given.

Description

"N-DEOXYRIBOSYLTRANSFERASES OF LACTOBACILLI, CORRESPONDING NUCLEOTIDE SEQUENCES AND THEIR

APPLICATIONS"

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

The analogues of nucleosides the structure of which comprises alterations of the sugar or heterocyclic base, form a family of molecules active in the treatment of numerous bacterial, viral, parasitic and fungal infections as well as in antitumour chemotherapy [P6rigaud et al., 1992]. Moreover the insecticidal and herbicidal properties of certain nucleoside antibiotics make them potential agents in the sector of agrichemicals and the environment [Isono, 1988]. The industry uses two methods for producing these analogues, organic synthesis and biocatalytic conversion (enzymatic conversion and microbiological conversion), which have advantages and, conversely, drawbacks. Organic synthesis makes it possible to achieve extremely widely varied chemical structures but requires multiple stages and is expensive in terms of reagents and solvents. On the other hand, the biocatalytic processes allow easy production in an aqueous medium but limited to a small number of possible compounds due to the specificity of the enzymes, which allow a limited range of analogues in the place of their physiological substrates. The phosphorylase nucleosides and N-deoxyribosyltransferase, which result from the purine and pyrimidine salvage pathways in the bacteria, are the enzymes most used for these enzymatic conversions (Krenisky et al., 1981).

There is therefore an urgent requirement to obtain enzymes for conversion of nucleosides and their derivatives, having a broadened enzyme activity in order to diversify the industrial production of these compounds. This is the technical problem which the inventors of the present invention propose to resolve.

The N-deoxyribosyltransferase of Lactobacillus leichmannii as well as that of L. helveticus, partially purified or not purified, is shown to be the best glycosyl group donor and tolerates a considerable number of structural variations on the base. This enzyme has been used for producing a certain number of analogues among which should be cited 2-chloro,2'-deoxyadenoside (Carson et al., 1984), 2',3'-dideoxynucleosides of natural bases (Carson and Wasson, 1988), or several pyrazolo(3,4-d)pyrimidine and derivatives of 2',3'-dideoxycytidine and the corresponding base (Fischer et al., 1990).

With the aim of making available recombinant enzymes capable of treating the widest possible variety of deviant substrates either by the base or by the sugar, the inventors have isolated genes coding for an Ndeoxyribosyltransferase activity of different strains of lactobacilli. This variety of N-deoxyribosyltransferase enzymes makes it possible to increase the chances of obtaining enzymes with specificity broadened by mutations in the wild-type genes or by chimeras of these wild-type genes.

Two classes of N-deoxyribosyltransferase have been distinguished (Danzin and Cardinaud, 1976), the first (Class I) designated ptd (for purine transdeoxyribosylase) catalyzing exclusively the exchange of deoxyribose between two purines: dR-Pur Pur' dR-Pur' Pur and the second (Class II) designated ntd (for nucleoside transdeoxyribosylase), 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 (Buck, 1997; dbjIBAA92683.21 (AB039914)).

Throughout the description and the claims of this specification the word "comprise" and variations of the word, such as "comprising" and "comprises" is not intended to exclude other additives, components, integers or steps.

A reference herein to a patent document or other matter which is given as prior art is not to be taken as an admission that that document or matter was known or that the information it contains was part of the common general knowledge as at the priority date of any of the claims.

A subject of the present invention is therefore an isolated or purified polypeptide of Lactobacillus having at least one Ndeoxyribosyltransferase activity with a sequence of amino acids chosen 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) coded by the ntd Lh gene of Lactobacillus helveticus.

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

According to a third embodiment, the polypeptide according to the invention is the N- W \Nge700000 7499971745\71745 page 3 oc deoxyribosyltransferase of SEQ ID No.6 coded by the ntd Lf gene of Lactobacillus fermentum.

According to a fourth embodiment, the polypeptide according to the invention is the Ndeoxyribosyltransferase of SEQ ID No.8 coded by the ntd gene of Lactobacillus crispatus.

According to a fifth embodiment, the polypeptide according to the invention is the Ndeoxyribosyltransferase of SEQ ID No.10 coded by the ntd gene of Lactobacillus amylovorus.

According to a sixth embodiment, the polypeptide according to the invention is the Ndeoxyribosyltransferase of SEQ ID No.12 coded 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 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; a polypeptide variant of a polypeptide with amino acid sequences defined in (c) a polypeptide homologous to the polypeptide defined in or and comprising at least 80% identity, preferably 85%, 87%, 90%, 95%, 97%, 98%, 99% identity with said polypeptide of a fragment of at least 15 consecutive amino acids, preferably 17, 20, 23, 40, 50, 100, 250 consecutive amino acids of a polypeptide defined in b) or and a biologically active fragment of a polypeptide defined in b) and 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 the two genes of the guaBA operon of which, which control the conversion of IMP to XMP then to GMP, as well as those of the deoCABD operon which control the degradation of the deoxynucleosides, have been deleted. In fact, these strains, in order to survive or grow, require a supply of deoxyguanosine (dR-G) to the culture medium and the presence of a N-deoxyribosyltransferase activity of a polypeptide according to the invention in order to carry out the exchange: dR-G A dR-A G.

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

By variant polypeptide is meant all of the mutated polypeptides which can exist naturally, in particular in the human being, and which correspond in particular to truncations, substitutions, deletions and/or additions of amino acid residues.

By homologous polypeptide is meant the 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 chimeric fusion. Among the homologous polypeptides, those are preferred, the amino acid sequence of which has at least 80% identity, preferably at least 85%, 87%, 90%, 93%, 95%, 97%, 98%, 99% identity with the amino acid sequences of the polypeptides according to the invention. In the case of a substitution, one or more consecutive or non-consecutive amino acids can be replaced by "equivalent" amino acids.

The expression "equivalent" amino acid is here intended to designate any amino acid capable of being substituted for one of the amino acids of the basic structure, without however modifying the characteristics or essential functional properties, such as their biological activities of deoxyribosyltransferase), corresponding polypeptides such as the in vivo induction of antibodies capable of recognizing the polypeptide the amino acid sequence of which is comprised in the amino acid 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 or one of its fragments. These equivalent amino acids can be determined either on the basis of their homology of structure with the amino acids for which they are substituted, or on the basis of the results of tests for cross-species reactivity to which the different polypeptides are capable of giving rise. By way of example, there will be mentioned the possibilities of substitutions capable of being carried out without resulting in a more profound modification of biological activities of the corresponding modified polypeptides, the replacements, for example of leucine by valine or isoleucine, of aspartic acid by glutamic acid, of glutamine by asparagine, of arginine by lysine etc., it being naturally possible to envisage the reverse substitutions 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 very acid environment, also form part of the invention.

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

Different protocols known to a person skilled in the art have been described for introducing mutations into the polypeptides. Among these, there should be mentioned by way of 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 stage is following by a stage of screening the mutant of interest i.e. in targeted a manner. In the latter case, the mutations are preferably introduced at the level of the catalytic site of the Ndeoxyribosyltransferases according to the invention.

Preferably a polypeptide according to the invention is a polypeptide constituted by the 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 or a sequence having at least preferably at least 85%, 90%, 95%, 98% and 99% identity with the 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 after optimal alignment. By polypeptide the amino acid sequence of which having a percentage identity of least preferably at least 85%, 90%, 95%, 98% and 99% after optimal alignment with a reference sequence, is meant the 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 the amino acid sequence of which has a percentage identity of least 80%, preferably at least 85%, 90%, 95%, 98% and 99% after optimal alignment with 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 or with one of their fragments according to the invention, the variant polypeptides coded by the variant peptide sequences as previously defined are preferred, in particular the polypeptides, the amino acid sequence of which has at least one mutation corresponding in particular to a truncation, deletion, substitution and/or addition of at least one amino acid residue relative to 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 or with one of their fragments; more preferably, the variant polypeptides having at least one mutation which reduces the specificity of the polypeptide according to the invention for its substrate, such that the variant polypeptides according to the invention are capable of catalyzing a larger variety of substrate, in order to obtain a wider range of base analogues.

The invention also relates to a purified or isolated polynucleotide characerized in that it codes for a polypeptide as defined previously and preferably for 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. Preferably, the polynucleotide according to the invention has the sequence SEQ ID No.l, 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 SEQ ID No.l, 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 sequence of a fragment of at least consecutive nucleotides, preferably at least 18, 21, 24, 27, 30, 35, 40, 50, 75, 100, 150, 200 consecutive nucleotides of 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; a nucleic sequence having a percentage identity of at least 70%, preferably at least 75%, 85%, 90%, 95%, 98%, 99% after optimal alignment with a sequence defined in a) or the complementary sequence or the RNA sequence corresponding to a sequence as defined in b) or c).

The polynucleotide according to the invention is also characterized in that its expression in the host cells, in particular the bacterial strains such as PAK6, make it possible to satisfy the guanine requirement of said strain. The PAK6 strain was deposited at the CNCM on 2nd May 2001 under No. 1-2664. The PAK6 strain corresponds to the bacterial strain of Escherichia coli MG 1655 deleted of two guaA and guaB genes, as well as of the deoC, deoA, deoB, deoD genes. The PAK6 strain (AguaBA::gm Adeo-ll) is auxotrophic for guanine in minimal glucose medium.

By nucleic acid, nucleic sequence or nucleic acid sequence, polynucleotide, oligonucleotide, polynucleotide sequence, nucleotide sequence, terms which will be used interchangeably in the present description, is meant a precise chain of nucleotides, modified or unmodified, making it possible to define a fragment or a region of a nucleic acid, comprising or not comprising non-natural nucleotides, and being able to correspond equally well to a double-stranded DNA, a single-stranded DNA and transcription products of said DNAs, and/or a fragment of

RNA.

It must be understood that the present invention does not relate to the nucleotide sequences in their natural chromosomal environment, i.e. in the natural state. They are sequences which have been isolated or purified, i.e. they have been collected, directly or indirectly, for example by copying, their environment having been at least partially modified. Thus the nucleic acids obtained by chemical synthesis are also meant.

By polynucleotide of complementary sequence is meant any DNA the nucleotides of which are 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 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 the orientation of which is reversed.

By "percentage identity" between two nucleic acid or amino acid sequences within the meaning of the present invention, is meant a percentage of nucleotides or amino acid residues identical 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 distributed at random and throughout their length. By "best alignment" or "significant alignment" is meant the alignment for which the percentage identity determined as follows is the highest. The comparisons of sequences between two nucleic acid or amino acid sequences are carried out in standard manner by comparing these sequences after having aligned them in significant manner, said comparison having been carried out by segment or by "window of comparison" in order to identify and compare the local regions of sequence similarity. The significant alignment of the sequences for the comparison can be carried out, apart from manually, by means of the local homology algorithm of Smith and Waterman (1981), by means of the local homology algorithm of Neddleman and Wunsch (1970), by means of the similarity search method of Pearson and Lipman (1988), by means of 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 the significant alignment, the BLAST program is 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 sequences aligned in significant manner, the nucleic acid or amino acid sequence to be compared being able to comprise additions or deletions relative to the reference sequence for a significant alignment between these two sequences. The percentage identity is calculated by determining the number of identical positions for which the nucleotide or the amino acid residue is identical between the two sequences, by dividing this number of identical positions by the total number of positions compared and by multiplying the result obtained by 100 in order to obtain the percentage identity between these two sequences.

By nucleic sequences having a percentage identity of at least 70%, preferably at least 75%, 80%, 85%, 98% and 99% after significant alignment with a reference sequence, is meant the nucleic sequences having, relative' to the reference nucleic sequence, certain modifications such as in particular a deletion, truncation, elongation, chimeric fusion, and/or a substitution, in particular a point substitution, and the nucleic sequence of which has at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, 98% and 99% identity after significant alignment with the reference nucleic sequence. These are preferably sequences the complementary sequences of which are capable of being hybridized 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 conditions of hybridization or of high stringency will be such that they ensure at least 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. A hybridization under conditions of high stringency signifies that the conditions of temperature and ionic force are chosen in such a manner that they allow the maintenance of the hybridization between two fragments of complementary DNA. By way of illustration, conditions of high stringency of the hybridization stage for the purposes of defining the polynucleotide fragments described above, are advantageously the following: the DNA-DNA or DNA-RNA hybridization is carried out in two stages: prehybridization at 420C for 3 hours in phosphate buffer (20 mM, pH 7.5) containing 5 x SSC (1 x SSC corresponds to a 0.15 M NaC1 0.015 M sodium citrate solution), 50% formamide, 7% sodium dodecyl sulphate (SDS), 10 x Denhardt's, 5% dextran sulphate and 1% salmon sperm DNA; hybridization per se for 20 hours at a temperature depending on the size of the probe 420C, for a probe of size 100 nucleotides) followed by two 20-minute washings at 200C in 2 x SSC 2% SDS, one washing at 200C in 0.1 x SSC 0.1% SDS. The last washing is carried out in 0.1 x SSC 0.1% SDS for minutes at 600C, for a probe of size 100 nucleotides). The hybridization conditions of high stringency described above for a polynucleotide of defined size, can be adapted by a person skilled in the art for oligonucleotides of a larger or smaller size, according to the teaching of Sambrook et al., 1989.

Among the nucleic sequences having a percentage identity of at least 70%, preferably at least 75%, 90%, 95%, 98% and 99% after significant alignment with the sequence according to the invention, the variant nucleic sequences of SEQ ID No.l, SEQ ID No.3, SEQ ID SEQ ID No.7, SEQ ID No.9, SEQ ID No.11, SEQ ID No.13 or their fragments are also preferred, i.e. all of the nucleic sequences corresponding to allelic variants, i.e. individual variations of the sequences SEQ ID No.l, SEQ ID No.3, SEQ ID No.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 is consituted by one of the sequences SEQ ID No.l, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID No.11, SEQ ID No.13, their complementary sequences or RNA sequences corresponding to SEQ ID No.l, 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 nucleic acid sequence according to the invention, also form part of the invention. Thus, the primers or probes according to the invention are useful for the detection, identification, assay or amplification of the nucleic acid sequence. In particular, they can make it possible to demonstrate or distinguish between the variant nucleic sequences, or to identify the genome sequence of new eukaryotic or prokaryotic genes, in particular bacterial, and more precisely, of Lactobacillus bacteria, coding for an Ndeoxyribosyltransferase, by 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 primers in processes for the detection, identification, assay or amplification of the nucleic sequence, have a minimal 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 primers and/or probes in processes implementing in particular the PCR (polymerase chain reaction) technique (Rolfs et al., 1991). This technique requires the choice of pairs of oligonucleotide primers framing the fragment which has to be amplified. Reference can, for example, be made to the technique described in US Patent No. 4,683,202. The amplified fragments can be identified, for example according to agarose or polyacrylamide gel electrophoresis, or according to a chromatographic technique such as gel filtration or ion exchange chromatography, then sequenced. The specificity of the amplification can be controlled by using as primers the nucleotide sequences of polynucleotides of the invention and as matrices, plasmids containing these sequences or 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 capable of being obtained by amplification using primers according to the invention.

Other techniques for amplification of the target nucleic acid can be advantageously used as an alternative to the PCR (PCR-like) using primer pairs of nucleotide sequences according to the invention. By PCR-like is meant any methods implementing direct or indirect reproductions of the nucleic acid sequences, or in which the marking systems have been amplified, these techniques being of course known. Generally, it is a matter of amplification of the DNA by a polymerase; when the original sample is an RNA, reverse transcription should be carried out beforehand. There are currently very numerous processes allowing this amplification, for example the SDA (Strand Displacement Amplification) technique (Walker et al., 1992), the TAS (Transcriptionbased Amplification System) technique described by Kwoh et al. (1989), the 3SR (Self-Sustained Sequence Replication) technique described by Guatelli et al.

(1990), the 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 RCR (Repair Chain Reaction) technique described by Segev (1992), the CPR (Cycling Probe Reaction) technique described by Duck et al. (1990), the Q--replicase amplification technique described by Miele et al. (1983). Certain 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 the implementation of an amplification reaction using primers according to the invention, or the implementation of a detection process using probes of the invention, a reverse transcriptase type enzyme in order 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 process according to the invention.

The probes hybridization technique can be carried out in various ways (Matthews et al. 1988). The most general method consists of immobilizing the nucleic acid extracted from the cells of different tissues or cells in culture on a support (such as nitrocellulose, nylon, polystyrene) in order to produce for example DNA chips, then incubating, under well defined conditions, the target nucleic acid immobilized with the probe. After the hybridization, the probe excess is eliminated and the hybrid molecules formed are detected by the appropriate method (measurement of the radioactivity, fluorescence or enzyme activity linked to the probe).

According to another method of using 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 using a second probe, called a "detection probe", marked by an easily detectable element.

Among the fragments of useful nucleic acids, there should moreover be mentioned in particular the anti-sense oligonucleotides, i.e. the structure of which ensures, by hybridization with the target sequence, an inhibition of the expression of the corresponding product. Sense oligonucleotides should also be mentioned, 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 can be marked directly or indirectly by a radioactive or non-radioactive compound, by methods well known to a person skilled in the art, in order to obtain a detectable and/or quantifiable signal. The nonmarked polynucleotide sequences according to the invention can be used directly as probes or primers.

The sequences are generally marked in order to obtain sequences which can be used for numerous applications. The marking 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, there can be mentioned 32 p, 33p, H, or 125I. The non-radioactive entities are selected from the ligands such as biotin, avidin, streptavidin, dioxygenin, haptens, colouring agents, luminescent agents such as the radioluminescent, chemoluminescent, bioluminescent, fluorescent, phosphorescent agents.

The invention also comprises a method for detection and/or assay of a polynucleotide according to the invention, in a biological sample, characterized in that it comprises the following stages: isolation of the DNA from the biological sample to be analyzed, or obtaining of a cDNA from the RNA of the biological sample; (ii) specific amplification of the DNA coding for the polypeptide according to the invention using primers; (iii) analysis of the amplification products. A subject of the invention is also 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 to carry out a DNA amplification reaction, and optionally (iii) a component making it possible to verify the sequence of the amplified fragment, more particularly a probe according to the invention.

The invention also comprises a method for detection and/or assay of a nucleic acid according to the invention, in a biological sample, characterized in that it comprises the following stages: bringing a polynucleotide according to the invention into contact with a biological sample; (ii) detection and/or assay of the hybrid formed between said polynucleotide and the nucleic acid of the biological sample. A subject of the invention is also 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 probe according to the invention, (ii) the reagents necessary for implementing a hybridization reaction, and/or, if appropriate, (iii) a pair of primers according to the invention, as well as the reagents necessary for a DNA amplification reaction.

Preferably, the biological sample according to the invention in which the detection and assay are carried out, is constituted by a culture medium, a cell homogenate, a body fluid, for example a human or animal serum, blood, milk.

The present invention also relates to the 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 a subject of the invention.

Preferably, the recombinant vectors according to the invention are: the vector called pLH2 comprising the polynucleotide SEQ ID No.1 as present in the bacterial strain deposited at the CNCM on 30th May 2001 under No.I- 2676; the pLH2 plasmid contains an Alu I fragment of 1.4 kb containing the gene coding for the type II Ndeoxyribosyltransferase of Lactobacillus helveticus CNRZ32 cloned in the SmaI site of the pBAM3 plasmid; the pLH2 plasmid, which expresses this enzyme, is propagated in the PAK6 strain which is auxotrophic for guanine; the vector called pLH4 comprising the polynucleotide SEQ ID No.3 as present in the bacterial strain deposited at the CNCM on 30th May 2001 under No.I- 2677; the pLH4 plasmid contains an Alu I fragment of 1.6 kb containing the gene coding for the type I Ndeoxyribosyltransferase of Lactobacillus helveticus CNRZ32 cloned in the SmaI site of the pBAM3 plasmid; the pLH4 plasmid, which expresses this enzyme, is propagated in the PAK6 strain which is auxotrophic for guanine; the vector called pLF6 comprising the polynucleotide SEQ ID No.5 as present in the bacterial strain deposited at the CNCM on 30th May 2001 under No.I- 2678; the pLF6 plasmid contains an Alu I fragment of 1.36 kb containing the gene coding for the type II N-deoxyribosyltransferase of Lactobacillus fermentum CIP102780T. The pLF6 plasmid, which expresses this enzyme, is propagated in the PAK6 strain which is auxotrophic for guanine; the vector called pLA comprising the polynucleotide SEQ ID No.11 as present in the bacterial strain deposited at the CNCM on 21st June 2001 under No.I- 2689; the pLA plasmid corresponds to the pSUl9 plasmid, at the sites PstI and BamHI an insert is cloned containing the gene coding for the type II Ndeoxyribosyltransferase of Lactobacillus acidophilus CNRZ 1296. The plasmid is propagated in the strain of Escherichia coli TG-I.

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

The different control signals are chosen as a function of the host cell used. To this end, the nucleic acid sequences according to the invention can be inserted into autonomous replication vectors inside the chosen host, or integrative vectors of the chosen host. Among the autonomous replication systems, as a function of the host cell, "plasmid", "cosmid", "phagemid" or "minichromosome" type systems or viral type systems are preferably used, the viral vectors being able in particular to be adenoviruses (Perricaudet et al., 1992), retroviruses, lentiviruses, poxviruses, or herpetic viruses (Epstein et al., 1992). A person skilled in the art knows the technologies which 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, for example, plasmidic or viral type systems; such viruses are, for example, the retroviruses (Temin, 1986), or the AAVs (Carter, 1993).

Among the non-viral vectors, naked polynucleotides such as naked DNA or naked RNA are preferred according to the technique developed by VICAL, the bacterial artificial chomosomes (BACs), yeast artificial chromosomes (YACs) for expression in yeast, mouse artificial chromosomes (MACs) for expression in murine cells and in preferred manner human artificial chromosomes (HACs) for expression in human cells.

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

The invention comprises moreover the host cells, in particular the eukaryotic and prokaryotic cells transformed by the vectors according to the invention.

Among the cells which can be used within the meaning of the present invention, bacteria and yeasts can be mentioned. According to a preferred embodiment of the invention, the bacterium is chosen from the group composed of Lactobacillus fermentum, Lactobacillus acidophilus, Lactobacillus amylovorus, Lactobacillus crispatus, Lactobacillus helveticum, Lactobacillus lactis, Escherichia coli, Bacillus subtilus, 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: the bacterium transformed by the pLH2 plasmid comprising the polynucleotide SEQ ID No.l, as deposited at the CNCM on 30th May 2001 under No.I- 2676; the bacterium transformed by the pLH4 plasmid comprising the polynucleotide SEQ ID No.3, as deposited at the CNCM on 30th May 2001 under No.I- 2677; the bacterium transformed by the pLF6 plasmid comprising the polynucleotide SEQ ID No.5, as deposited at the CNCM on 30th May 2001 under No.I- 2678; -the bacterium transformed by the pLA plasmid comprising the polynucleotide SEQ ID No.11, as deposited at the CNCM on 21st June 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, there should also be mentioned the cells of insects, animal or plant cells.

Preferably, the cell according to the invention is free from any enzyme activity capable of degrading said precursor deoxyribonucleoside or said deoxyribonucleoside obtained by bioenzymatic reaction catalyzed by a polypeptide according to the invention. Alternatively, said host cell can be free from additional bioenzymatic activities intended to transform the precursor deoxyribonucleoside and/or deoxyribonucleoside obtained by the bioenzymatic reaction catalyzed by the polypeptide according to the invention. Among these additional bioenzymatic activities, there should be mentioned phosphorylation, sulphatation, acetylation, succinylation, methylation.

The nucleic acid sequence coding for the Ndeoxyribosyltransferases according to the invention is either naturally present in said cell or is introduced into said cell by the recombinant DNA techniques known to a person 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. By heterologous nucleic acid sequence is meant a nucleic acid sequence which is not naturally present in the cell according to the invention.

The present invention also relates to metazoic, plant or animal organisms, preferably mammals, except humans, comprising one of said cells transformed according to the invention. Among the animals according to the invention, rodents are preferred, in particular mice, rats or rabbits, expressing at least one polypeptide according to the invention.

The cells, preferably bacterial, or fungal, in particular of yeast, as well as the metazoic organisms according to the invention can be used 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 are cultured, in particular the cells of the present invention, under conditions allowing the expression and optionally the secretion of a recombinant polypeptide coded by a nucleic acid sequence according to the invention, and said recombinant polypeptide is recovered. The recombinant polypeptides capable of being obtained by this production method also form part of the invention. They can be presented in glycosylated or nonglycosylated form, and may or may not have the tertiary structure of the natural protein. The recombinant polypeptide sequences can also be modified in order to improve their solubility, in particular in aqueous solvents. Such modifications are known to a person skilled in the art such as for example the deletion of hydrophobic domains or the substitution of hydrophobic amino acids by hydrophilic amino acids.

These polypeptides can be produced from the nucleic acid sequences defined above, according to the techniques for producing recombinant polypeptides known to a person skilled in the art. In this case, the nucleic acid sequence used is placed under the control of signals allowing its expression in a host cell.

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

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

The polypeptides according to the present invention can also be obtained by chemical synthesis using one of the numerous known peptide syntheses, for example the techniques using solid phases or techniques using partial solid phases, by condensation of fragments or by a synthesis in standard solution. The polypeptides obtained by chemical synthesis and being able to 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 one of the subjects of the present invention to provide a monoclonal or polyclonal antibody and its fragments, characterized in that they selectively and/or specifically bind a polypeptide according to the invention. The chimeric antibodies, humanized antibodies and single-chain antibodies also form part of the invention. The antibody fragments according to the invention are preferably Fab, F(ab')2, Fc or Fv fragments. The polyclonal antibodies can be prepared, for example by immunization of an animal, in particular a mouse, with a polypeptide according to the invention combindd with an immune response adjuvant, then purification of the specific antibodies contained in the serum of the animals immunized on an affinity column on which the polypeptide having served as antigen has been fixed beforehand. The polyclonal antibodies according to the invention can also be prepared by purification on an affinity column on which a polypeptide according to the invention has been immobilized beforehand. The monoclonal antibodies can advantageously be prepared from hybridomas according to the technique described by Kohler and Milstein in 1975.

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-deoxyribosyltransferase polypeptide.

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

Moreover, apart from their use for the purification of the 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 can also be marked in the same manner as described previously for the nucleic probes of the invention and in preferred manner with an enzymatic, fluorescent or radioactive marking.

The antibodies of the invention also constitute a means of analysis of the polypeptide expression according to the invention, for example by immunofluorescence, marking with gold, enzyme immunoconjugates. More generally, the antibodies of the invention can 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 or immunohistochemistry or in "western" blotting experiments. Thus, a process for detecting a polypeptide according to the invention in a biological sample, comprising the stages of bringing the biological sample into contact with an antibody according to the invention and demonstrating the antigen-antibody complex formed is also a subject of the invention.

It is also one of the subjects of the present invention to provide a process for in vitro or in vivo enzymatic synthesis of deoxyribonucleotides characterized in that it comprises at least one reaction stage catalyzed by at least one N-deoxyribosyltransferase according to the invention. The process according to the invention is characterized in that said Ndeoxyribosyltransferase 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 composed of the purines bound by N9, pyrimidines bound by N1, azines bound by N1, imidazoles bound by N1, said second nucleobases being able to carry substitutions of the hydrogens at the non-bound positions. Preferably, said second nucleobase is selected from the group composed of 6-methyl purine, 2-amino-6methylmercaptopurine, 6-dimethylaminopurine, azacytidine, 2,6-dichloropurine, 6-chloroguanine, 6chloropurine, 6-aza-thymine, 5-fluoro-uracile, ethyl-4carboxylate, imidazole-4-carboxamide and 1,2,4-triazole-3-carboxamide.

Said first nucleobase is itself preferably selected from the group composed of adenine, guanine, thymine, uracile and hypoxanthine. These lists are not exhaustive, and it is evident that natural or non-natural analogues of nucleobases can be used in the present invention as substrate of an N-deoxyribosyltransferase of the invention.

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

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

Optionally, the in vivo process according to the invention is characterized in that it moreover comprises the stage of introducing into the host cell the first nucleobase present in a deoxyribonucleoside and the second nucleobase simultaneously and/or one after the other.

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

Alternatively, these deoxyribonucleosides capable of being produced in large quantities and inexpensively by the biosynthesis method according to the invention constitute herbicides and insecticides.

The present invention also provides a process for nutritional screening intended to isolate deoxyribosyltransferases, preferably the polypeptides according to the invention but also their homologues or their mutants. This first screening according to the invention comprises the stages: (optionally) obtaining a bacterial strain, such as Escherichia coli, auxotrophic for guanine.

Preferably this strain is incapable of growing in the presence of deoxyguanosine as a source of guanine. In preferred manner, it is the PAK 6 strain.

(ii) transfer of exogenous DNA, preferably in the form of an expression vector, into the bacterium, the exogenous DNA being capable of comprising a sequence coding for a deoxyribosyltransferase.

(iii) culture of the bacteria obtained in Stage (ii) on a medium containing deoxyguanosine.

(iv) isolation of the exogenous DNA transferred into the bacteria of Stage (iii) which have developed on the medium containing deoxyguanosine.

The present invention also provides a nutritional screening for distinguishing the deoxyribosyltransferase I and II activities, preferably in particular for distinguishing between the ntd and ptd polypeptides according to the invention. This second screening comprises the stages of: obtaining a bacterial strain such as for example Escherichia coli, auxotrophic for guanine and thymidine. Preferably, this strain is incapable of growing in the presence of guanine and thymidine. In preferred manner, it is the PAK 26 strain (A ,guaBguaA Adeo-11 AthyA erm A (udp-metE)zif9 TnlO) is auxotrophic for methionine, guanine and thymidine.

(ii) transfer of the exogenous DNA, preferably in the form of an expression vector, into the bacterium, the exogenous DNA being capable of comprising a sequence coding for a deoxyribosyltransferase I or II.

(iii) culture of the bacteria obtained in Stage (ii) on a medium containing deoxyguanosine, then determination of whether the bacteria are growing or not. If the bacteria are growing, then the exogenous DNA codes for a deoxyribosyltransferase II activity which is expressed in said bacterium. If the bacteria are not growing, then the exogenous DNA is capable of coding for a deoxyribosyltransferase I activity.

Other characteristics and advantages of the invention are clear from the rest of the description, with examples represented hereafter.

EXAMPLES

1. MATERIAL AND METHODS 1.1 Strains and culturing conditions The strains of lactic bacteria used originate from the CNRZ (Centre National de Recherche Zootechnique) collection, Unit6 de Recherches Laitieres et G4n6tique Appliquee, INRA, Jouy en Josas. They are cultured in MRS medium (from Man et al., J. Appl. Bacteriol., 23: 130- 135, 1960) and incubated at 30°C, 37 0 C or 42 0 C according to the species. The Escherichia coli TG1 strain, provided by Stratag6ne, is cultured in LB (Luria broth base lOg/L, Agar-agar 16g/L) under agitation and at 37°C.

1.2. Preparation of total cellular DNA of lactic bacteria: The cultures at the end of the exponential phase are centrifuged for 5 minutes at 13,000 g. The pellet corresponding to a culture of 2 ml is taken up in 200 pl of TES (50 mM Tris, pH8, 10 mM EDTA, pH8, 250 mM saccharose) containing 20 pg/ml of lysozyme and 50 U/ml of mutanolysine (Sigma). After an incubation of one hour at 37°C, the clarification of the preparation is obtained by adding 60 pl of 20% SDS.

The extraction of the nucleic acids is carried out by adding to the lysate 500 pl of saturated phenol in water, pH8, to which 0.1% hydroxyquinoline and 100 pl of a mixture of isoamylic chloroform-alcohol (24/1, V/V) has been added. The solution is homogenized then centrifuged for 10 minutes at 13,000 g and at ambient temperature.

The upper, limpid phase containing the nucleic acids is retained. The extraction is repeated three times on the latter in order to eliminate the undesired cellular constituents. The phenol traces are eliminated by adding 500 pl of isoamylic chloroform-alcohol to the aqueous phase. After homogenization and centrifuging for 3 minutes at 15,000 g and at 4°C, the nucleic acids contained in the upper aqueous phase are precipitated by the addition of a volume of cold isopropanol. After an incubation of one hour at -200C, a centrifugation is carried out for 20 minutes at 15,000 g and at 4°C. The isopropanol is eliminated and replaced by 500 pl of ethanol. A final centrifugation of 10 minutes at 15,000 g and at 4°C allows a pellet of nucleic acids to be recovered. This is left to dry in an evaporator and resuspended in 200 pl of sterile water containing 10 pl of RNase at 10 pg/pl. After 15 minutes of incubation at 37°C to agitate the enzyme degrading the RNAs, 10 pl of the DNA solution is migrated by electrophoresis in an 0.8% agarose gel in order to evaluate the concentration and the quality.

1.3. Polymerase chain reaction of DNA (PCR): Polymerase chain reactions (PCR) are carried out in a reaction volume of 100 pl containing 20 to 100 ng of DNA, 0.5 uM primers, 200 pM dNTPs (dATP, dCTP, dGTP, dTTP) in a 10 mM Tris-HCl buffer pH 9, 50 mM KC1, 1.5 mM MgCl 2 0.002% BSA as well as 2.5 units of Taq polymerase.

Thirty amplification cycles were used (Gene Amp PCR systems 2400, Perkin Elmer). The inventors defined two ntdl (SEQ ID no. 15) and ntd2 primers (SEQ ID no. 16) starting from the ntd sequence of Lactobacillus leichmanii described by Huck (personal communication): ntdl 5'-AGA CGA TCT ACT TCG GTG- 18 bases Tm= 54 0

C

3' ntd2 5'-ACG GCA CCT TCG TAG AAG- 18 bases Tm= 56 0

C

3' 1.4. Southern-type hybridization: Enzymatic restriction of the DNAs. The total DNAs are digested by one or more restriction enzymes. The enzymes used are: BamHI, BglII, ClaI, EcoRI, HindIII, HpaI, NcoI, NotI, PstI, XbaI, XhoI (Bio-Lab). Digestion is carried out in a final volume of 40 pl containing 70 U of enzyme, 4 ul of 10X NEB buffer (Bio-Lab) and 4 to 8 pg of DNA. The incubation is carried out for 1 hour minutes at 37 0

C.

Transfer of the DNA to a membrane. The total DNA fragments resulting from the enzymatic digestion are separated using a 0.7% agarose gel. After migration, the agarose gel is placed under agitation in a depurination solution (0.25N HC1) for 30 minutes. This process thus allows the transfer of DNA fragments larger than 10 kbs.

After rinsing with water, the DNA is denatured by placing the gel for 40 minutes into a solution of 5M NaC1, NaOH. The gel is rinsed with water then incubated again for 30 minutes in a neutralization solution, 1.5M NaCl, Tris HC1; pH 7.5. The DNAs are transferred by capillarity onto a positively charged nylon membrane (Hybond Amersham). They are eluted by a rising flux of 20X SSC (0.3M trisodium citrate; 3M NaCl; pH7). After the transfer, the DNAs are covalently bound onto the membrane using a UV Stratalinker 2400 apparatus (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) in order to eliminate the PCR primers. The necessary concentration of the probe is ng/pl. The DNA is marked using the ECL marking kit (Amersham). To do this, the DNA is denatured by heating for 5 minutes at 100 0 C and immediately recooled in ice. A volume of marking reagent (peroxidase) then a volume of glutaraldehyde solution are added. This solution is incubated for 10 minutes at 37 0 C for covalently binding the peroxidase to the DNA.

Hybridization and development. After a prehybridization of one hour at 420C in hybridization buffer, the membrane is hybridized for 16 hours at 42 0

C

in the presence of the marked probe. In order to eliminate the probe bound in non-specific manner, the membrane is washed for 20 minutes at 420C in two successive baths of buffer: 6M urea 0.4% SDS SSC, then rinsed for 5 minutes in two successive baths of buffer: 0.3M sodium citrate 3M NaCi pH 7. The development is carried out by autoradiography according to the protocol of the ECL kit. A first development reagent containing hydrogen peroxide is reduced by peroxidase bound to the probe. Then the luminol contained in a second development reagent is oxidized, producing light which exposes the autoradiographic film.

1.5. Cloning the PCT fragments: The homologous ntd genes amplified by PCR are inserted into the plasmid vector pBluescript II SK+ of E.

coli. TG1 (Stratagene). This plasmid is first restricted in its single site by the EcoRV enzyme (Gibco-BRL) which creates free ends. The digestion mixing is carried out in a volume of 30 pl, containing 4 pl of DNA. The DNA fragments amplified to be cloned should have their 5' and 3' ends free in order to allow cloning. The preparation of the free ends of 50 pl of PCR products purified using the Wizard kit (Promega) is carried out in a reaction volume of 100 pl containing 3.6 units of polymerase DNA of the T4 phage (Bio-Lab) and 6 units of polymerase I DNA (or Klenow fragment) (Bio-Lab), not having a 5' 3' exonucleic activity. The polymerization is carried out for 20 minutes at 11 0 C then the enzymes are deactivated after 10 minutes at 75 0 C. The DNA is then precipitated in the presence of two volumes of 100% ethanol, glycogen and 3M sodium acetate, pH 4.8. The mixture is placed for one hour at -20 0 C then centrifuged for 20 minutes at 15,000 g. The pellet is rinsed with 250 ul of ethanol, centrifuged again for 10 minutes at 15,000 g, dried in an evaporator and resuspended in 20 pl of sterile water.

The DNA restricts the pBS-SK+ plasmid and the amplified fragment is comigrated on a 0.7% agarose gel in order to evaluate their respective concentrations: the number of molecules of the fragment to be cloned should be three to four times greater than that of the plasmid.

The ligation is carried out in a volume of 10 ul containing 60 ng of insert, 26 ng of restricted pBS-SK+ plasmid, 2 units of ligase (T4 DNA ligase, Boehringer- Mannheim), overnight at 16 0 C. The ligation products are dialyzed on a 0.025 pm millipore filter so as to eliminate the salts and to thus avoid electric arcs during electroporation.

1.6. Transformation: Preparation of electroconpetent cells of the TG1 strain of E. coli. Starting from 5 ml of an overnight culture at 37 0 C under agitation, 500 ml of LB medium are inoculated. The culture is placed under agitation at 37°C until an O.D.

600 om 1 is reached. It is then recooled for 2 hours in ice then cold-centrifuged for 10 minutes at 5,000 rpm. The supernatant is eliminated, the pellet is taken up in 400 ml of cold water. This preparation is cold-centrifuged for 10 minutes and at 5,000 rpm. The pellet obtained is taken up again in 250 ml of cold water. Following a centrifugation of 10 minutes, the pellet is taken up in 25 ml of cold water then the cells are suspended in a final volume of 1 ml of 10% glycerol, and aliquotted before being rapidly frozen in liquid nitrogen.

Transformation by electroporation and selection of clones. The electrocompetent cells preserved at -80 0 C are thawed in ice then brought into contact with 5 pl of ligature mixture in an electroporation flask. The Gene- Pulser (Bio-Rad) is regulated at 200 volts, 25 mF, 250 ohms. The cells are then subjected to electroporation. 1 ml of an SOC solution is added (20g/L bactopeptone, yeast extract, 2ml/L 5M NaC1, 2.5 ml/L 1M KC1, 10 ml/L 1M MgCl2, 10 ml/L 1M MgSO 4 containing 0.4% glucose in cell suspension which is incubated at 37 0 C for one hour. The cells are then plated on an LB selective medium -Xgal bromo-4-chloro-3-indolyl-P-D-galactoside, lpg/ml) -IPTG (isopropythio-p-D-galactoside, lpg/ml) -ampicillin pg/ml) and incubated at 37 0 C overnight.

Rapid extraction of plasmidic DNA of recombinant E.

coli clones by alkaline lysis. The E. coli cells transformed and cultivated in LB medium containing ampicillin (100 pg/ml) are harvested by centrifugation at 15,000 for 10 minutes at 4 0 C. They are resuspended in 100 p1 of a 50 mM saccharose solution, 25 mM Tris-HCl, pH 8, mM EDTA, pH 8. The alkaline lysis and the denaturation of the DNA is carried out by addition of 200 p1 0.2N NaOH, 1% SDS. The reaction medium is left for 1 minute at ambient temperature after having added 200 p1 of chloroform. Then 150 p1 of a solution of 5M potassium acetate, glacial acetic acid are added. The reaction medium is centrifuged for 15 minutes at 13,000 g at 4 0

C.

The aqueous phase containing the DNA is precipitated in the presence of 2 volumes of 100% ethanol then centrifuged for 20 minutes at 13,000 g at 4 0 C. The pellet is washed in 70% ethanol, centrifuged for 10 minutes at 13,000 g then resuspended in 30 p1 of sterile water containing RNase at 10 ng/ml.

1.7. Inverse PCR Inverse PCR allows the regions flanking a fragment of DNA of known sequence to be amplified. This technique takes place in three steps: Digestion of the DNA matrix. The DNA matrix is digested by one or two restriction enzymes chosen such that they do not cleave in the known gene sequence and they allow a fragment of suitable size (I to 3 kb) to be obtained. To choose a suitable enzyme, the total DNA is digested separately by several enzymes. Then Southerntype hybridizations are carried out using the DNA fragment of known sequence as a probe. The digestions for which the probe hybridizes with a fragment of 1 to 3 kb are Used for the inverse PCR. The DNA fragments obtained by digestion are circularized. For this purpose, 100 units of T4 DNA ligase and 100 p of ligation buffer are added to 4 to 8 pg of DNA in a final volume of 1 ml. The ligation mixture is incubated at 15 0 C overnight. The ligated DNA is then precipitated with 100 pl of 3M sodium acetate, pH 4.8, 700 pl of isopropanol and 2 pl of glycogen, then centrifuged for 30 minutes at 13,000 g at 4°C. The pellet is rinsed in 300 pl of 70% ethanol and centrifuged for 10 minutes at 13,000 g at 4°C. The pellet is taken up in 25 pl of ultrapure water.

Amplification of circularized DNAs using different primers. The polymerase chain reactions are carried out in a reaction volume of 100 pl containing 20 to 100 ng of DNA, 0.5 pM primers, 200 pM dNTPs (dATP, dCTP, dGTP, dTTP) in a 10 mM Tris-HCl buffer pH 9, 50 mM KC1, MgCI 2 with 1.5 mM BSA at 0.002% and 2.5 units of Taq polymerase.

The amplification is carried out under the following conditions: 940°C 3 minutes 940°C 30 seconds to 60°C (according to the 25 cycles Tm of the primers used) 1 minute 72°C 3 minutes (Gene Amp PCR systems 2400, Perkin Elmer).

Sequencing. The PCR fragments are purified by the Wizard kit (Promega) in order to eliminate the nonincorporated oligonucleotides, the salts and the Taq polymerase. The sequencing is carried out 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 of the primers. The sequence reactions are carried out in a reaction volume of 20 pl containing 30 ng of DNA, 4 pl of DyeT Mix (Perkin Elmer Biosystems) and 0.1 mM of primer.

Cycle: 96 0 C 1 minute 96 0 C 10 seconds 5 seconds 25 cycles 60°C 4 minutes pl of 3M sodium acetate, pH 4.6, 50 pl of ethanol and 1 pl of glycogen are added to each sequence reaction. The solution is left for 15 minutes at ambient temperature then centrifuged for 20 minutes at 13,000 g.

The pellet is then rinsed with 250 pl of 70% ethanol then centrifuged for 10 minutes at 13,000 g. The pellet is then taken up in 6 pl of sequence blue (83% formamide, 8.3 mM EDTA, 0.5% dextran blue 2,000,000 (Sigma)). The samples are denatured for 3 minutes at 90% and 3 pl deposited on 4% acrylamide gel.

2. A SINGLE NUTRITIONAL SCREENING FOR THE TWO CLASSES OF N-DEOXYRIBOSYLTRANSFERASE IN ESCHERICHIA COLI A functional screening allowing the production of guanine to be selected was established on E. coli by deleting the two genes of the guaBA operon which controls the conversion of IMP into XMP then into GMP and also those of the deoCABD operon which controls the degradation of deoxynucleosides to give the PAK 6 strain.

The E. coli genome specifies an activity allowing the base G to be converted into GMP (guanine phosphoribosyltransferase encoded by the gpt gene), and also an activity allowing the base G to be released from the dR-G deoxynucleoside (purine nucleoside phosphorylase encoded by the deoD gene in the deo operon). The PAK 6 strain thus has a requirement for guanine which cannot be satisfied by the addition of deoxyguanosine The use of deoxyguanosine (dR-G) can however be selected if an N-deoxyribosyltransferase activity is expressed in the PAK6 strain in order to carry out the exchange: dR-G A dR-A G.

This exchange between two purine bases can be catalyzed by the two enzyme classes. In fact, the introduction of the ntd gene of L. leichmannii into the PAK 6 strain allows the requirement for guanine to be satisfied using deoxyguanosine (dR-G) and adenine 3. FUNCTIONAL CLONING OF THE PDT GENE OF L.

HELVETICUS.

DNA fragments of a size comprised between 1 and 2kb obtained by partial digestion (AluI) of L. helveticus CNRZ 32 were ligated in a ColE1 plasmid (of pUC type digested by Smal and desphosphorylated) and the mixture is used to transform the PAK6 strain. The transforming clones were selected in a glucose mineral medium to which deoxyguanosine (dR-G) and adenine have been added to the final concentration of 0.3 mM.

One of the transforming clones proved to propagate a plasmid containing an insert controlling a Class I Ndeoxyribosyltransferase activity and deviating from the restriction profile of the ntd gene of L. helveticus. The sequence of this insert develops a gene specifying a polypeptide of 167 amino acids with a molecular weight of 18790.70 Daltons having a similarity of 28.6% with the NTD polypeptide of L. leichmanii. The sequence of this gene called ptd deviates from that of the ntd genes making them impossible to hybridize identity).

Incidentally the ntd gene of L. helveticus controlling a Class II N-deoxyribosyltransferase activity could be isolated once again among the transforming clones selected.

4. FUNCTIONAL CLONING OF THE NTD GENE OF L.

ERMENTUM.

The same nutritional cloning and selection operations were carried out starting from genomic DNA of the L. fermentum CIP 102980T strain. The transforming clones selected proved to propagate a plasmid the inserts of which, having similar restriction profiles, controlled a Class II N-deoxyribosyltransferase activity. The sequence of one of these inserts developed a gene specifying a polypeptide of 168 amino acids with a molecular weight of 18878.20 Daltons having a similarity of 32.9% with the NTD polypeptide of L. leichmanii and 36.7% with the PTD polypeptide of L. helveticus. The sequence of this gene deviates from that of the ntd and ptd genes which makes them impossible to hybridize. The NTD polypeptide of L. fermentum has a more marked evolutive relationship for the enzyme of Class I (PTD of L. helveticus) and a functional affinity to Class II, suggesting an early evolutive divergence in the evolution of these enzymes. Its enzymatic activity could prove to be different to those of other species and be suitable for the preparation of a very large spectrum of nucleosides.

INVERSE PCR CLONING OF FOUR NTD GENES.

Using degenerated oligonucleotides starting from regions of the amino acid sequence of the NTD polypeptide of L. leichmanii (Htck, 1997) an internal fragment on the ntd gene of L. helveticus was amplified. Starting from this fragment, oligonucleotides were synthesized so as to obtain all the gene by inverse PCR.

Starting from the two ntd sequences of L.

leichmannii and L. helveticus, we redefined the consensus primers by isolating the ntd genes from other species of lactobacilli such as L. acidophilus, L. crispatus, L.

amylovorus with the same result as that described above.

6. A nutritional screening to distinguish the two activities of deoxyribosyltransferase I and II.

To distinguish between the two deoxyribosyltransferase activities, the plasmidic DNA of different selected colonies was extracted then used to transform the auxotrophic PAK 26 strain for guanine and thymidine. In the PAK 26 strain, the dTMP cannot be synthesized starting from dUMP because the thymidylate synthase encoded by the thyA gene has been deactivated.

Moreover, the thymine cannot be a source of thymidine because the thymidine phosphorylase encoded by the deoA gene and uridine phosphorylase encoded by the udp gene have been deleted. Deoxyguanosine (dR-G) and thymine (T) will be the sources of guanine and thymidine only if an N-deoxyribosyltransferase II activity is expressed in the PAK 26 strain to catalyze the exchange reaction dG T dT G. Only the colonies expressing an Ndeoxyribosyltransferase II activity can grow on a mineral glucose medium supplemented by deoxyguanosine and thymine as sources of guanine and thymidine. This second screening for example allowed the Ndeoxyribosyltransferase II (ntd) activity to be correlated with the pLH2 plasmid containing the polynucleotide of SEQ ID No. 1 and coding for the ntd enzyme of lactobacillus helveticus and the Ndeoxyribosyltransferase I (ptd) activity with the pLH4 plasmid containing the polynucleotide of SEQ ID No. 3 and coding for the ptd enzyme of lactobacillus helveticus.

Table 1: Growth of the PAK 6 strain expressing or not expressing an N-deoxyribosyltransferase activity on a glucose mineral medium (in vivo) and enzymatic activity of corresponding raw extracts (in vitro) in in vivo vitro A G dG dG A dC T dG A dC +A PAK 6 pSUl9 ntd 6 PAK L1 ntd 6 PAK Lh ptd 6 PAK Lh ntd 6 PAK growth absence of growth PAK 6: MG1655 AguaBA::Apra, Adeo ntd L1: gene coding for the N-deoxyribosyltransferase of Lactobacillus leichmannii ntd Lh: gene coding for the N-deoxyribosyltransferase of Lactobacillus helveticus ntd Lf: gene coding for the N-deoxyribosyltransferase of Lactobacillus fermentum ptd Lh: gene coding for the purine deoxyribosyltransferase of Lactobacillus helveticus A: adenine; G: guanine; T: thymine; dG: deoxyguanosine; dC: deoxycytidine

REFERENCES

Cadwell et al. (1992) Research 2 28-33.

Carson Wasson (1988) Biochem. Biophys. Res. Comm. 155, 829-834.

Carson et al., (1990) Proc. Natl. Acad.Sci. USA 81, 2232- 2236.

Carter (1993) Curr. Op. Biotechnology 3, 533.

Danzin Cardinaud (1976) Eur. J. Biochem. 62, 365-372.

Duck et al. (1990), Biotechniques, 9, 142.

Epstein (1992) M6decine/Sciences, 8, 902.

Fischer et al., (1990) Ger. Offen. DE 3840160.

Guatelli et al. (1990), Proc. Natl. Acad. Sci. USA 87: 1874.

Isono (1988) J. Antibiotics 12, 1711-1739.

Kievitis et al., (1991) J. Virol. Methods, 35, 273.

K8hler et Milstein. (1975) Nature 256, 495.

Krenitsky et al., (1981) Biochemistry 20, 3615-3621.

Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA, 86, 1173.

Landegren et al. (1988) Science 241, 1077.

Matthews et al. (1988) Anal. Biochem., 169, 1-25.

Miele et al., (1983) J. Mol. Biol., 171, 281-285.

Neddleman et Wunsch (1970) J. Mol. Biol. 48 443 Pearson et Lipman (1988) Proc. Natl. Acad. Sci. USA 2444 P6rigaud et al., (1992) Annales de l'Institut Pasteur Actualites 3, 179-215.

Perricaudet et al. (1992), La Recherche 23 471.

Rolfs, A. et al. (1991), Berlin Springer-Verlag.

Sambrook et al. (1989) Molecular cloning a laboratory manual second edition Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY. USA Segev, (1992), Kessler C. Springer Verlag, Berlin, New York, 197-205.

46 Smith et Waterman (1981) Ad. App. Math. 2 482 Temin, (1986) Retrovirus vectors for gene transfer. In Kucherlapati ed. Gene Transfer.

Walker et al. (1992) EMBO J. 1 945-951 Wilson et al. (1995) Synthesis 1465-1479.

Wong C.H. et al. (1995) Angew. Chem. Int. Ed. Engl. 34, 412-432 and 521-546.

Claims (18)

1. Process for in-vivo or in-vitro enzymatic synthesis of deoxyribonucleotides comprising at least one reaction stage catalyzed by one N-deoxyribosyltransferase of Lactobacillus, which comprises an amino acid sequence at least 90% identical to SEQ ID NO.2.

2. Process for enzymatic synthesis of deoxyribonucleotides comprising at least one reaction stage catalyzed by one N-deoxyribosyltransferase of Lactobacillus comprising the amino acid sequence SEQ ID NO.2.

3. Process according to claim 1 or 2, wherein the expression of said N-deoxyribosyltransferase in the PAK6 strain deposited at the CNCM on May 2 nd 2001, under No.I-2664, makes it possible to satisfy the guanine requirement of said strain.

4. Process according to any one of claims 1 to 3, wherein said N-deoxyribosyltransferase is encoded by the ntd gene of Lactobacillus helveticus. Process according to any one of claims 1 to 4, wherein said N-deoxyribosyltransferase catalyzes the exchange of a first nucleobase present in a deoxyribonucleoside by a second nucleobase.

6. Process according to claim 5, wherein said second nucleobase is selected from the group composed of the purines bound by N9, pyrimidines bound by N1, azines bound by N1, imidazoles bound by N1, said second W: \NgeA700000 740999717495\717495 Claims Aug 07doc nucleobases being able to carry substitutions of the hydrogens at the non-bound positions.

7. Process according to claim 6, wherein said second nucleobase is selected from the group composed of 6- methyl purine, 2-amino-6-methylmercaptopurine, 6- dimethylaminopurine, 5-azacytidine, 2,6-dichloropurine, 6- chloroguanine, 6-chloropurine, 6-aza-thymine, uracile, ethyl-4-amino-5-imidazole carboxylate, imidazole- 4-carboxamide and 1,2,4-triazole-3-carboxamide.

8. Process according to claim 7, wherein said first nucleobase is selected from the group composed of adenine, guanine, thymine, uracile and hypoxanthine.

9. Process for in-vivo enzymatic synthesis of deoxyribonucleotides according to any one of claims 5 to 8, further comprising the stage of introducing into a host cell expressing said N-desoxyribosyltransferase of Lactobacillus, the first nucleobase present in a deoxyribonucleoside. Process according to claim 9, further comprising the stage of introducing into said host cell, the second nucleobase present in a deoxyribonucleoside.

11. Process according to claim 10, further comprising the stage of introducing into said host cell, the first nucleobase present in a deoxyribonucleoside and the second nucleobase simultaneously and/or staggered over time. W \NigeA7000D 74999\M717495\717495 Claims Aug 07doc 49 S12. Process according to any one of claims z 1 to 8, for in-vitro enzymatic synthesis of 00 deoxyribonucleotides.

13. Process for preparing a recombinant 1 polypeptide, wherein a host cell is transformed by: C a cloning or expression vector comprising a IO c purified or isolated polynucleotide from Lactobacillus Sencoding a N-deoxyribosyltransferase, which comprises an C 10 amino acid sequence at least 90 identical to SEQ ID NO.2, or pLH2 vector comprising SEQ ID NO.1, as deposited at the CNCM on 30 th May 2001 under No.I-2676; is cultured under conditions allowing the expression, and optionally the secretion, of said recombinant polypeptide, and recovering said recombinant polypeptide.

14. Recombinant polypeptide capable of being obtained by a process according to claim 13. An isolated polypeptide having at least one N-deoxyribosyltransferase activity comprising an amino acid sequence at least 90 identical to SEQ ID NO.2.

16. An isolated polypeptide comprising the amino acid sequence SEQ ID NO.2.

17. An isolated polypeptide according to claims 15 or 16, which makes it possible to satisfy the guanine requirement of the PAK6 strain deposited at the CNCM on May 2 nd 2001, under No.I-2664. W:%NigeA7D000 749999W71749517495 Cla,s Nov O7doc 0 18. Purified or isolated polynucleotide encoding 00 a polypeptide as defined in any one of claims 14 to 17.

19. Purified or isolated polynucleotide according to claim 18, wherein the sequence of said C polynucleotide comprises SEQ ID NO.1. Use of a polynucleotide according to claims C A 10 18 or 19, as a primer for the amplification or polymerization of nucleic acid sequences of N-desoxyribosyltransferases.

21. Use of a polynucleotide according to claims 18 or 19, as a probe for the detection of nucleic acid sequences of N-desoxyribosyltransferases.

22. Recombinant cloning and/or expression vector comprising a polynucleotide according to claims 18 or 19, or expressing a polypeptide according to any one of claims 14 to 17.

23. Recombinant vector called pLH2 comprising the polynucleotide SEQ ID No.1 as present in the bacterial strain deposited at the CNCM on 30th May 2001 under No.I-

2676. 24. Host cell, that is transformed by the recombinant vector according to claim 23. Bacterium transformed by the recombinant vector pLH2 comprising the polynucleotide SEQ ID No.1, as W:\NQe%7DDODOO 499M7i495i7l7495 Claims Nov 07..doc deposited at the CNCM on 30th May 2001 under No.I-2676. 26. Metazoic, plant or animal organism, except human comprising a cell according to claim 24. 27. Monoclonal or polyclonal antibody or a fragment thereof, which binds selectively a polypeptide according to one of claims 14 to 17. W \Nge70000 71499M717496177495 Caaas Aug o.Doc WO 03/025163 PCT/FRO2/03120 LISTE DE SEQUENCES <110> Institut PASTEUR Institut National de la Recherche Agronoimique INRA <120> N-desoxyribosyl transferases de Lactobacillus, sequences nucleotidiques correspondantes et leurs applications <130> D19532 <160> 16 <170> Patentln Ver. 2.1 <210> 1 <211> 477 <212> ADN <213> Lactobacillus helveticus NTD <220> <221> CDS <222> (477) <400> 1 atg aac aag aaa aag act tta tat ttt ggt gcc ggt tgg ttt aat gaa 48 Met Asn Lys Lys Lys Thr Leu Tyr Phe Gly Ala Gly Trp Phe Asn Glu 1 5 10 sag caa aac aaa gct tac aaa gas gca atg gca gct tta aaa gaa aat 96 Lys Gin Asn Lys Ala Tyr Lys Glu Ala Met Ala Ala Leu Lys Glu Asn 25 cca aca gtt get tta gaa aat agt tat gtg ccc ctt gaa. aac caa tac 144 Pro Thr Val Asp Leu Glu Asn Ser Tyr Val Pro Leu Glu Asn Gin Tyr 40 aag ggt att cgc att gat gaa cat cca gaa tac ttg cac aac att gaa 192 Lys Gly Ile Arg Ile Asp Glu His Pro Giu Tyr Leu His Asn Ile Giu 55 tgg gct tct gca acc tac cac sat gat tta gta gga att aag act tct 240 Trp Ala Ser Ala Thr Tyr His Asn Asp Leu Val Gly Ile Lys Thr Ser 70 75 gat gtc atg ctt ggc gta tat ttg cca gaa gsa gaa gac gtc ggc tta 288 Asp Val Met Leu Gly Val Tyr Leu Pro Giu Glu Glu Asp Val Gly Leu 90 ggc atg gaa ctg ggc tac gca tta tct caa gga asa tat att tta ttg 336 Gly Met Glu Leu Gly Tyr Ala Leu Ser Gin Gly Lys Tyr Ile Leu Lee 100 105 110 gtt atc cca gat gaa gat tac ggc aag cca etc eec tta atg sgc tgg 384 Val Ile Pro Asp Glu Asp Tyr Giy Lys Pro Ile Asn Leu Met Ser Trp 115 120 125 ggc gtt tgt gac aat qcc atc aeg etc egt gaa tte aea gac ttc gac 432 Gly Val Cys Asp Asn Ala Ile Lys Ile Ser Glu Leu Lys Asp Phe Asp 130 135 140 WO 03/025163 PCT/FR02/03120 ttt aac aag cct cgc tac aat ttc tac gac gga get gta tat taa 477 Phe Asn Lys Pro Arg Tyr Asn Phe Tyr Asp Gly Ala Val Tyr 145 150 155 <210> 2 <211> 158 <212> PRT <213> Lactobacillus helveticus NTD <400> 2 Met Asn Lys Lys Lys Thr Leu Tyr Phe Gly Ala Gly Trp Phe Asn Glu 1 5 10 Lys Gin Asn Lys Ala Tyr Lys Glu Ala Met Ala Ala Leu Lys Glu Asn 25 Pro Thr Val Asp Leu Glu Asn Ser Tyr Val Pro Leu Glu Asn Gin Tyr 40 Lys Gly Ile Arg Ile Asp Glu His Pro Glu Tyr Leu His Asn Ile Glu 55 Trp Ala Ser Ala Thr Tyr His Asn Asp Leu Val Gly Ile Lys Thr Ser 70 75 Asp Val Met Leu Gly Val Tyr Leu Pro Glu Glu Glu Asp Val Gly Leu 90 Gly Met Glu Leu Gly Tyr Ala Leu Ser Gin Gly Lys Tyr Ile Leu Leu 100 105 110 Val Ile Pro Asp Glu Asp Tyr Gly Lys Pro Ile Asn Leu Met Ser Trp 115 120 125 Gly Val Cys Asp Asn Ala Ile Lys Ile Ser Glu Leu Lys Asp Phe Asp 130 135 140 Phe Asn Lys Pro Arg Tyr Asn Phe Tyr Asp Gly Ala Val Tyr 145 150 155 <210> 3 <211> 504 <212> ADN <213> Lactobacillus helveticus PTD <220> <221> CDS <222> <400> 3 atg aaa gca gta gtt cca aca gga aaa att tat tta ggc tca cca ttt 48 Met Lys Ala Val Val Pro Thr Gly Lys Ile Tyr Leu Gly Ser Pro Phe 1 5 10 tac age gat get caa aga gaa aga gca got aag gca aaa gag ttg tta 96 Tyr Ser Asp Ala Gin Arg Glu Arg Ala Ala Lys Ala Lys Glu Leu Leu 25 WO 03/025163 gca aaa aat cta agc atc gcg cac gtc ttc ttc ccc ttt Ala Lys Asn Leu Ser Ilie Ala His Val Phe Phe Pro Phe PCT/FR02/03120 ttc aco gat Phe Thr Asp atg gtt tgg Met Val Trp aat gcc act Asn Ala Thr ggC tet gc Gly Ser Ala atc ttg gtg Ile Leu Val 115 ctg atg atc Leu Met Ile 130 ttt gaa aaa Phe Giu Lys 145 40 iat cct gaa att ggc ggc isn Pro Glu Ile Gly Gly :ac caa aat gat tta act 'yr Gin Asn Asp Leu Thr .ta tat gat aty gat caa .eu Tyr Asp Met Asp Gin tc atg cgt gcg atg cat 'he Met Arg Ala Met His 105 at ccc gaa aaa gaa aag 'is Pro Glu Lys Giu Lys 20 125 cc acc atc att gat ggc 'hr Thr Ile Ile Asp Gly 140 ac ttc aac gaa tgt cct .sn Pha Asn Glu Cys Pro gat Asp atc Ile ggt Gly tta Leu aag Lys 110 aaa Lys aat Asn ttt Phe: gtt cgc ggt tac ggt atc tat taa Val Arg Gly Tyr Gly Ile Tyr 165 <210> 4 <211> 167 <212> ERT <213> Lactobacilius <400> 4 Met Lys Ala Val Val 1 5 Tyr Ser Asp Ala Gin Ala Lys Asn Leu Ser Phe Thr Asp Pro Asp Met Val Trp Arg Asp Asn Ala Thr Cys Gly heiveticus PTD Pro Thr Gly Lys Arg Glu Arg Ala 25 Ile Ala His VTal Giu Lys Asn Pro Ala Thr Tyr Gin Val Phe Lou Tyr Ser Pro Giu Leu Asp Asp Ile Arg Gly Ile Lou Asp WO 03/025163 PCT/FR02103120 Gly Scr Ala Phe Giu Ile Gly Phe Met Arg Ala Met His Lys Pro Va. 100 105 110 Ile Leu Val Pro Phe Thr Glu His Pro Glu Lys Glu Lys Lys Met Asn 115 120 125 Leu Met Ile Ala Gin Gly Val Thr Thr Ile Ile Asp Gly Asn Thr Giu 130 135 140 Phe Giu Lys Leu Ala Asp Tyr Ase Phe Asn Giu Cys Pro Phe Asn Pro 145 150 155 160 Val Arg Gly Tyr Gly Ile Tyr 165 <210> <211> 516 <212> ADN <213> Lactobacilius fermentum NTD <220> <221> CDS <222> (504) <400> ttg aaa ast ace gac cca gtt get az Leu Lys Asn Thr Asp Pro Val Ala As 1 5 C act ass att tac ctg ;n Thr Lys Ile Tyr Leu get ac Ala Thr age Ser caa Gin ttC Phe ggC Gly gte Val gac Asp ect Pro tte Phe gas Giu tat Tyr ct C Leu act Thr gga Gly gtt Val 115 sac Asn gee Ala na Lys ga Giu tee Ser att Ile 100 tta Leu ga Glu sac As n gat Asp tgg Trp gat Asp tgt Cys eta Leu gsa esa egt Giu Gin Arg ceg set gte Pro Thr Val 40 gca cge gta Ala Arg Val ens att gee Gin Ile Ala 70 gte tge gtt Val Cys Val atg gaa ate Met Giu Ile ect ttt set Pro Phe Thr 120 gee egg ggt Ala Arg Gly gce ege Ala Arg 25 gge gtt Gly Val gee tee Asp Ser act tee Thr Tyr get tta Ala Leu 90 gge ntg Gly Met 105 sag aaa Lys Lys ate cet Ile Pro gtt ec Val His gat cet Asp Pro aat ase Asn Asn 75 tee get Tyr Asp tte gte Phe Val gat ang Asp Lys ens get Gin Ala eng cen Gin Pro gee ggc Ala Gly gac etc Asp Leu atg gee Met Asp gee ete Ala Leu 110 tct get Ser Ala 125 eta gee Leu Ala tte gat Phe Asp gte ttt Val Phe aee geg Asn Ala eas att Gin Ile eat ens His Lys tat ga Tyr Giu 96 144 192 240 288 336 384 432 get aee eta atg eta Ala Asn Leu Met Leu gta aet sec t99 ttg gaa eet aat Val Thr Thr Trp Leu Glu Pro Ace WO 03/025163 WO 03/25163PCT/FR02/03120 130 gac ttt agt ccc tta aaa Asp Phe Ser Pro ILeu Lys 145 150 cct ttc cca cca ttc aag Pro Phe Pro Pro Phe Lys 165 140 ttt aac ttt aac cac cca atg gct caa Phe Asn Phe Asn His Pro Met Ala Gin 155 160 ttc taactaacct aa Phe <210> 6 <211> 168 <212> PPT <213> Lactobacillus fermentui NTD <400> 6 Leu Lys 1 Ser Phe Gin Leu Phe Gin Gly Ser Val Gly Asp Glu_ Pro Ile Ala Asn 130 Asp Phe 145 Pro Phe Asni Phe Glu Tyr Leu Thr Gly Val 115 Leu S er Pro Thr Asn Al a Lys Glu Ser Ile 100 Leu Met Pro Pro Asp 5 Glu Asn Asp Trp Asp Cys Leu Leu Leu Phe 165 Pro Glu Pro Al a Gin 70 Val Met Pro Ala Lys 150 Lys Val Ala Gin Arg Thr Val 40 Arg Val 55 Ile Ala Cys Val Giu Ile Phe Thr 120 Arg Gly 135 Asp Phe Val Phe Asn Thr Lys 10 Ala Arg Ile 25 Gly Val Val Asp Ser Asp Thr Tyr Asn Ala Leu Tyr 90 Gly Met Phe 105 Lys Lys Asp Val Thr Thr Aen Phe Asn 155 Ile Pro His Pro Asn Asp Val Lys Trp 140 His Tyr Gin Gin Ala Asp Met Al a Ser 125 Leu Pro Leu Ala Ala Leu Pro Phe Gly Vai Leu Asn Asp Gin Leu His 110 Ala Tyr Gie Pro Met Ala Thr Ala Asp Phe Al a Ile Lys Glu Asn Gin 160 <210> 7 <211> 255 <212> ADN <213> Lactobacilius crispatus NTD <220> <221> CDS <222> (254) WO 03/025163 PCT/FR0203120 <400> 7 ac aec cag tac aag ggt atc cgc gtt gat gaa cac oct gaa tao ttg 47 Asn Gin Tyr Lys Gly Ile Arg Val Asp Glu His Pro Glu Tyr Leu 1 5 10 cac gac att gaa tgg goa tea got acc tac cat aao gac tta gta ggg His Asp Ile Glu Trp Ala Ser Ala Thr Tyr His Asn Asp Leu Val Gly 25 att aag toc agc gac ato atg ott ggc gtt tao ttg cot gaa gaa gaa 143 Ile Lys Set Set Asp Ile Met Leu Gly Val Tyr Leu Pro Glu Giu Glu 40 gat gtt ggt ctg gga atg gaa ctt ggc tat goc ctt te aaa ggc aag 191 Asp Val Sly Leu Gly Met Giu Leu Sly Tyr Ala Leu Set Lys Gly Lys 55 tac atc ttg ttg gta att cot gat gaa gat tao ggt aeg cca atc aac 239 Tyr Ile Leu Leu Val Ile Pro Asp Giu Asp Tyr Gly Lys Pro Ile Asn 70 tta atg ago tgg ggc a 255 Leu Met Set Trp Gly <210> 8 <211> 84 <212> PRT <213> Lactobacillus orispatus NTD <400> 8 Asn Gin Tyr Lys Gly Ile Arg Val Asp Glu His Pro Giu Tyr Leu His 1 5 10 Asp Ile Giu Trp Ala Set Ala Thr Tyr His Asn Asp Leu Val Giy Ile 25 Lys Ser Set Asp Ile Met Leu Gly Val Tyr Leu Pro Giu Glu Giu Asp 40 Val Sly Leu Gly Met Glu Leu Gly Tyr Ala Leu Ser Lys Gly Lys Tyr 55 Ile Leu Leu Val Ile Pro Asp Giu Asp Tyr Gly Lys Pro Ile Asn Leu 70 75 Met Ser Trp Gly <210> 9 <211> 399 <212> ADN <213> Lactobacillus amylovorus NTD <220> <221> CDS <222> WO 03/025163 <400> 9 atg gaa got tta aag nag aac coct Met Giu Ala Leu Lys Lys Asn Pro 1 5 act gtt gac tta gaa Thr Val Asp Leu Glu PCT/FR02/03121 aac agt tao Asn Ser Tyr 10 gtC Val gas Glu tta Leu gaa Glu caa Gin oca Pro agc Ser gat Asp cca ctt Pro Leu tat tta Tyr Leu gtt ggt Val Gly gaa gaa Glu Glu ggt aaa Gly Lys ato aao lie Asn gan ttg Glu Leu 115 ggt got Gly Ala 130 gat Asp cac His att Ile gat Asp tao Tyr ttg Leu aaa Lys gto Val aac Asn gao Asp aag Lys gtt Val ato Ile atg Met gao Asp tat Tyr caa Gin att Ile tct Ser ggo Gly 70 ttg Leu ago Ser tto Phe tao Tyr gaa Glu ton Ser 55 ott Leu ott Leu tgg Trp gao Asp aaa ggo Lys Gly 25 tgg gca Trp Ala 40 gao gta Asp Val ggg atg Gly Met gto ato Val Ile ggC gtt Gly Val 105 ttt aao Phe Asn 120 ato cgo Ile Arg ton tct Ser Ser atg otc Met Leu gaa ctt Glu Leu 75 cct gao Pro Asp 90 tgo gao Cys Asp aga cct Arg Pro gtt Val acc Thr ggt Gly ggo Gly gaa Glu aao Asn cgo Arg gat Asp tao Tyr gtt Val tao Tyr gao Asp gtn Vai tto Phe 125 gaa Glu cac His tat Tyr gca Ala tat Tyr ato Ile 110 aac I ksn I cac His nat Asa tta Leu ttg Leu ggt Gly sag Lys tt Phe cca Pro gao Asp cct Pro tct Ser aag Lys ato Ile tao Tyr 48 96 144 192 240 288 336 384 <210> <211> 133 <212> PRT <213> Lactobaillus <400> Met Glu Ala Leu Lys 1 5 Val Pro Leu Asp Asn Glu Tyr Leu His Asp Leu Val Giy Ile Lys Glu Glu Giu Asp Val Gin Gly Lys Tyr Ile amylovorus NTD Lys Asn Pro Thr Val 10 Gin Tyr Lys Gly Ile 25 Ile Giu Trp Ala Ser 40 Ser Ser Asp Val Met 55 Gly Leu Gly Met Glu 70 Leu Leu Val Ile Pro 90 Leu Glu Val Asp Thr Tyr Gly Vai Gly Tyr Glu Asp Asn Ser Tyr Glu His Pro His Asn Asp Tyr Leu Pro Ala Leu Ser Tyr Gly Lys WO 03/025163 PCT/FR0203120 Pro Ile Asn Leu Met Ser Trp Gly Val Cys Asp Asn Val Ile Lys Ile 100 105 110 Ser Giu Leu Lys Asp The Asp Phe Ass Arg Pro Arg Phe Asn Phe Tyr 115 120 125 Asp Gly Ala Val Tyr 130 <210> 12 <211> 480 <212> ADN <213> Lactobacilius acidophilus NTD <220> <221> CDS <222> <400> 11 atg atg gca aaa aca aaa act tta tat Met Met Ala Lys Thr Lys Thr Leu Tyr ttC Phe ggc gct ggt tgg ttt aat Gly Ala Gly Trp Phe Asn gas Glu sac Asn tat Tyr gaa Glu tca Ser ctt Leu ctc Leu tgg Trp gac Asp 145 caa aat Gin Asn act gtt Thr Val gat att Asp Ile gca tct Ala Ser stt atg Ile Met stg gas Met Giu 100 att cct Ile Pro 115 gta tgt Val Cys aat sag Asn Lys sag Lys gat Asp cgt Arg gct Ala tta Leu ctt Leu gac Asp gat Asp cca Pro get Ala ttg Leu gtt Vai act Thr 70 ggg Gly ggc Gly gaa Glu sac Asn cgc Arg 150 tat Tyr gas Glu gat Asp tat Tyr gtt Val tac Tyr gat Asp gct Ala 135 ttt Phe aaa Lys sat Asn 40 gas Glu cac His tac Tyr gca Ala tat Tyr 120 att Ile sac Asn gcs Ala 25 agt Ser cat His sac Asn tta Leu tta Leu 105 ggc Gly sag Lys gct Ala tat Tyr cct Pro gac Asp cct Pro 90 tca Ser sag Lys atc Ile atg Met gtt Val gaa Glu tts Leu 75 gaa Glu caa Gin cct Pro agc Ser gas Glu cca Pro tac Tyr stt Ile gas Glu ggc Gly atc Ile gas Glu 140 gct Ala ctt Leu tta Leu ggt Gly gaa Glu aaa Lye sac Asn 125 ttg Leu tta Leu gaa Glu cac His ate Ile gat Asp tat Tyr 110 ttg Leu sag Lys aaa Lys aat Asn gac Asp aaa Lye gtt Val atc Ile atg Met gac Asp caa Gin cas Gin att Ile tct Ser ggt Gly tta Leu agt Ser ttc Phe taa 160 96 144 192 240 288 336 384 432 480 ttc tat gat Phe Tyr Asp 155 ggC gct gta tat Gly Ala Val Tyr WO 03/025163 PCT/FR02/03120 <210> 12 <211> 159 <212> PRT <213> Lactobacillus acidophilus <400> 12 Met Met Ala Lys Thr Lys Thr Leu 1 5 Glu Lys Gin Asn Lys Ala Tyr Lys Asn Pro Thr Val Asp Leu Glu Asn 40 Tyr Lys Asp Ile Arg Val Asp Clu 55 Glu Trp Ala Ser Ala Thr Tyr His 70 Ser Asp Ile Met Leu Gly Val Tyr Leu Gly Met Glu Leu Gly Tyr Ala 100 Leu Val Ile Pro Asp Glu Asp Tyr 115 120 Trp Gly Val Cys Asp Asn Ala Ile 130 135 Asp Phe Asn Lys Pro Arg Phe Asn 145 150 NTD Tyr Phe Gly Ala Gly Trp Phe Asn 10 Ala Ala Met Giu Ala Leu Lys Gln 25 Ser Tyr Val Pro Leu Giu Asn Gln His Pro Giu Tyr Leu His Asp Ile 60 Asn Asp Leu Ile Gly Ile Lys Ser 75 Leu Pro Giu Giu Giu Asp Val Gly 90 Leu Ser Gin Gly Lys Tyr Ile Leu 105 110 Gly Lys Pro Ile Asn Leu Met Ser 125 Lys lie Ser Giu Leu Lys Asp Phe 110 Phe Tyr Asp Gly Ala Val Tyr 155 <210> 13 <211> 795 <212> ADN <213> Lactobacillus heiveticus NTD <220> <221> CDS <222> (140)..(616) <220> <223> n signifie n'importe quel nuci~otide a, g, c ou t/u <400> 13 aaaaaaattt tcagtattag tcattgaatt ttaccttcca ttatggaett actattttta gcgtaagtta acaagacgtt tttttcaatc gaaaatatgt taaagttaat tcgtcagcaa 120 tttttatggq ganaaaatt atg aac aag aaa aag act tta tat ttt ggt gac 172 Met Asn Lys Lys Lys Thr Leu Tyr Phe Gly Ala 1 5 WO 03/025163 ggt tgg ttt Gly Trp Phe gct tta aaa Ala Leu Lys aat gaa Asn Giu gsa aat Glu Asn aag aa Lys Gin eca a Pro Thr sac aaa Ass Lys 20 gtt gat Val Asp tac aaa Tyr Lys gsa sat Glu Asn PCT/FR02/03120 ges atg gea Ala Met Ala tat gtg ccc Tyr Val Pro 35 ctt ga Leu Glu aac caa tac aag ggt att egc att Asn Gin Tyr Lys ttg cac sac att gaa tgg Leu gga Gl gaa Giu aaa Lys aac As n tta Leu 140 His att Ile ga c Asp tat Tyr tta Leu 125 aa Lys Asn aag Lys gte Val att Ile 110 atg Met gac Asp Ile act Thr gge Gly tta. Leu age Ser ttc Phe Glu Trp 65 tot gat Ser Asp tta g Leu Gly ttg gtt Lau Val tgg ggc Trp Gly gae ttt Gly Ile 50 got tct Ala Ser gte atg Val Met atg- ga Met Giu ate oca Ile Pro 115 gtt tgt Val Cys- 130 sac sag Arg gca Al a ott Leu etg Lau 100 gat Asp gao Asp zct le ace Thr ggo Gly 85 9gc Gly ga Glu sat Asn ego gat Asp tao Tyr 70 gta Val tao! Tyr gat Asp gee Al a tac gas Glu, 050 His tat Tyr (j a Al a tac Tyr ate Ile 135 sat. cat His at Asn ttg Lau tts Leu Gly 120 aag Lys tte eea Pro gat Asp Pro tot Ser 105 sag Lys ate Ile tac gas Glu tta Leu gas Glu csaa Gin eca Pro agt Ser gar! Asp tao Tyr gta Val gas Glu 9ga Gly ate Ile gas Glu gga 316 364 412 460 508 556 604 656 Asp Phe Asn Lys Pro Arg Tyr Asn Phe Tyr 150 155 get gta tat tas aaaataagca aSctaaatga octategctt aaaaattgeg Ala Val Tyr ataggteatt ttttaatatt atetgtcatg tatassatct ttcttaataa atatacteca agtgattttc caaaaaastt attattotat aceacttoa tatggaagtc Cgagtcaett atgtaaatca tatateact <210> 14 <211> 158 <212> PRT <213> Lactobacilius helveticus NTD <400> 14 Met Asn Lys Lys Lys Thr Leu Tyr Phe Gly Ala Gly Trp Phe Asn Giu 1 5 10 Lys Gin Asn Lys Ala Tyr Lys Giu Ala Met Ala Ala Leu Lys Glu Asn 25 Pro Thr Val Asp Leu Glu Asn Ser Tyr Val Pro Leu Giu Asn Gin Tyr 40 10/11 WO 03/025163 Lys Gly Ile Trp Ala Ser Asp Val Met Gly Met Glu Val Ie Pro 115 Gly Val Cys 130 Phe Asn Lys 145 Arg Ile Asp Ala Thr Tyr 70 Leu Gly Val Leu Gly Tyr 100 Asp Glu Asp Asp Asn Ala Pro Arg Tyr 150 Glu 55 His Tyr Al a Tyr Ile Asn His Asn Leu Leu Gly 120 Lys Phe Pro Asp Pro Ser 105 Lys Ile Tyr. Glu Leu Giu 90 Gin Pro Ser Asp Tyr Val 75 Glu Gly Ile Glu Gly 155 Leu His Gly Ile Glu Asp Lys Tyr Asn Leu 125 Leu Lys 140 Ala Val PCT/FRO2/03120 Asn Ile Giu Lys Thr Ser Val Gly Leu Tie Leu Leu 110 Met Ser Trp Asp Phe Asp Tyr <210> <211> 18 <212> ADN <213> Lactobacillus leichmannii NTDI <400> agacgatcta cttcggtg <210> 16 <211> 18 <212> ADN <213> Lactobacillus ieichniannii NTD2 <400> 16 acggcacctt cgtagaag 11/11