AU2008201222A1 - N-deoxyribosyltransferase of lactobacilli, corresponding nucleotide sequences and their applications - Google Patents

Description

AUSTRALIA

Patents Act COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Class Application Number: Lodged:

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00q 0O 0 rs Complete Specification Lodged: Accepted: Published: Priority Related Art: Name of Applicant: Institut Pasteur and Institut National de la Recherche Agronomique Actual Inventor(s): Rachel Cotaya, Pierre-Alexandre Kaminski, Philippe Marliere, Pascal Quenee, Patrick Taillez Address for Service and Correspondence: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Invention Title: N-DEOXYRIBOSYLTRANSFERASE OF LACTOBACILLI, CORRESPONDING NUCLEOTIDE SEQUENCES AND THEIR APPLICATIONS Our Ref: 824973 POF Code: 11188/329842, 465301 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): 00 0 "N-DEOXYRIBOSYLTRANSFERASE OF LACTOBACILLI, CORRESPONDING 0 NUCLEOTIDE SEQUENCES AND THEIR APPLICATIONS" (t SThe present application is a divisional application from Australian Patent Application No. 2002362377, the entire disclosure of which is incorporated herein by reference.

The present invention relates to the field of biology, and C- more particularly to the microbiological production of base Sanalogues. The present invention relates to new polypeptides

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00 and their fragments, isolated from Lactobacillus, having at least one N-deoxyribosyltransferase 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 [Perigaud 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 W rk Wic amn\700000.799999 \717495\717495p l 2 00 compounds due to the specificity of the enzymes, which 0 0 allow a limited range of analogues in the place of their

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physiological substrates. The phosphorylase nucleosides Sand 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 00 10 derivatives, having a broadened enzyme activity in order Sto 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

-I

3 in the wild-type genes or by chimeras of these wild-type genes.

00 O Two classes of N-deoxyribosyltransferase have been C-q 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: C(N dR-Pyr Pur dR-Pur Pyr 00 SdR-Pyr Pyr' dR-Pyr' Pyr (N dR-Pur Pur' dR-Pur' Pur.

Only two genes specifying Class II enzymes, designated ntd, have been reported to date (Buck, 1997; dbjJBAA92683.21 (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- WWpnef700000 7491M 717495717495 pQe 3.doc 00 deoxyribosyltransferase of SEQ ID No.6 coded by the ntd 0 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.

C- According to a fifth embodiment, the polypeptide c- C- according to the invention is the Ndeoxyribosyltransferase of SEQ ID No.10 coded by the ntd 00 10 gene of Lactobacillus amylovorus.

0 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 00 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 Sstrains, 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 C- polypeptide according to the invention in order to carry C out the exchange: dR-G A dR-A G.

0 In the present description, the term polypeptide 00 10 will be used equally to designate a protein or a peptide.

O 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), 6 00 corresponding polypeptides such as the in vivo induction C of antibodies capable of recognizing the polypeptide the amino acid sequence of which is comprised in the amino C acid sequence SEQ ID No.2, SEQ ID No.4, SEQ ID No.6, SEQ S 5 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 c- determined either on the basis of their homology of c-i (M structure with the amino acids for which they are 0 substituted, or on the basis of the results of tests for 00 10 cross-species reactivity to which the different Spolypeptides 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 0 activity. The variant polypeptide, the homologous C 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 Sby way of example, the polymerase chain reaction (PCR) in 00 10 the presence of manganese (Cadwell et al., 1992). The Smutations 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 00 alignment with the sequences SEQ ID No.2, SEQ ID No.4, SSEQ 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 Sinvention, the variant polypeptides coded by the variant t 5 peptide sequences as previously defined are preferred, in particular the polypeptides, the amino acid sequence of -q which has at least one mutation corresponding in C-i particular to a truncation, deletion, substitution and/or Saddition of at least one amino acid residue relative to 00 10 the sequences SEQ ID No.2, SEQ ID No.4, SEQ ID No.6, SEQ SID 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 00 nucleotides of the sequence SEQ ID No.1, SEQ ID No.3, SEQ SID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID No.11, SEQ ID No.13; a nucleic sequence having a percentage Sidentity 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 ,q sequence or the RNA sequence corresponding to a sequence Sas defined in b) or c).

The polynucleotide according to the invention is 00 10 also characterized in that its expression in the host Scells, 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 00 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 Sacids 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 C- No.l, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, (Ml SEQ ID No.11, SEQ ID No.13 or part of SEQ ID No.l, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID 00 10 No.ll, 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 00 algorithms (GAP, BESTFIT, BLAST P, BLAST N, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison,

WI)

SIn 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.

q 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, 00 SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ C ID No.11, SEQ ID No.13 of the invention. Preferably, the specific conditions of hybridization or of high C stringency will be such that they ensure at least S 5 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 c-i other. A hybridization under conditions of high 0 stringency signifies that the conditions of temperature 00 10 and ionic force are chosen in such a manner that they 0 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 NaCl 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 42 0 C, for a probe of size 100 nucleotides) followed by two 20-minute washings at 20 0 C 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.

13 00 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 C- No.13 or their fragments are also preferred, i.e. all of Cq the nucleic sequences corresponding to allelic variants, 0 i.e. individual variations of the sequences SEQ ID No.l, 00 10 SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ 0 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.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, 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 00 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, Spreferably 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.

(CN The polynucleotides according to the invention can Sthus be used as primers and/or probes in processes 00 10 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 00 meant any methods implementing direct or indirect reproductions of the nucleic acid sequences, or in which N cthe marking systems have been amplified, these techniques Sbeing 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 Sexample the SDA (Strand Displacement Amplification) O 10 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-P-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.

00 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 Sextracted 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 in excess is eliminated and the O 10 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.

17 00 The probes, primers and oligonucleotides according to the invention can be marked directly or indirectly by Sa 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.

C The sequences are generally marked in order to obtain sequences which can be used for numerous 00 10 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, 33 p, 3H, 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 00 possible to verify the sequence of the amplified fragment, more particularly a probe according to the invention.

SThe 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 00 10 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.l as present in the bacterial strain 00 deposited at the CNCM on 30th May 2001 under No.I- O 2676; the pLH2 plasmid contains an Alu I fragment of 1.4 kb containing the gene coding for the type II N- Sdeoxyribosyltransferase 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.ll as present in the bacterial strain deposited at the CNCM on 21st June 2001 under No.I- 2689; the pLA plasmid corresponds to the pSU19 plasmid, at the sites PstI and BamHI an insert is cloned containing the gene coding for the type II Ndeoxyribosyltransferase of Lactobacillus acidophilus 00 CNRZ 1296. The plasmid is propagated in the strain Sof Escherichia coli TG-I.

The vectors according to the invention comprise the Selements necessary for the expression and in particular, S 5 preferably a promoter, of the translation initiation and termination signals, as well as appropriate regions for -sq regulation of the transcription. They must be able to be maintained in stable fashion in the cell and can Soptionally have particular signals specifying the 00 10 secretion of the translated protein.

SThe 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 00 cells and in preferred manner human artificial 0 chromosomes (HACs) for expression in human cells.

Such vectors are prepared according to the methods Scurrently 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 .q lipofection, electroporation, heat shock, transformation after chemical permeabilization of the membrane, cell Sfusion.

00 10 The invention comprises moreover the host cells, in Sparticular 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 00 deposited at the CNCM on 30th May 2001 under No.I- 0 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; S- the bacterium transformed by the pLA plasmid (q comprising the polynucleotide SEQ ID No.11, as Sdeposited at the CNCM on 21st June under No.I-2689; 00 10 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 00 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 C 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 00 skilled in the art such as for example the deletion of hydrophobic domains or the substitution of hydrophobic amino acids by hydrophilic amino acids.

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

00 10 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 OO techniques using solid phases or techniques using partial D solid phases, by condensation of fragments or by a synthesis in standard solution. The polypeptides obtained Sby 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.

SIt is therefore also one of the subjects of the present 00 10 invention to provide a monoclonal or polyclonal antibody Sand 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 0 physiological function of said N-deoxyribosyltransferase 0 polypeptide.

The invention also relates to methods for the Sdetection 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 q in that they are obtained by a method according to the Sinvention.

O 10 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.

00 It is also one of the subjects of the present C invention to provide a process for in vitro or in vivo enzymatic synthesis of deoxyribonucleotides characterized Sin that it comprises at least one reaction stage catalyzed by at least one N-deoxyribosyltransferase according to the invention. The process according to the Cq invention is characterized in that said NeC deoxyribosyltransferase catalyzes the exchange of a first O nucleobase present in a deoxyribonucleoside by a second 00 10 nucleobase.

SAccording 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.

00 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 Snucleobase 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 q nucleobase present in a deoxyribonucleoside and the second nucleobase simultaneously and/or one after the OO 10 other.

SThe 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 00 form of an expression vector, into the C bacterium, the exogenous DNA being capable of t comprising a sequence coding for a deoxyribosyltransferase.

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

C- (iv) isolation of the exogenous DNA transferred into the bacteria of Stage (iii) which have developed cO 10 on the medium containing deoxyguanosine.

00 The present invention also provides a nutritional Ci 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-ll 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 0 deoxyribosyltransferase II activity which is expressed in said bacterium. If the bacteria are CI not growing, then the exogenous DNA is capable Sof coding for a deoxyribosyltransferase

I

activity.

CI Other characteristics and advantages of the invention are clear from the rest of the description, with examples CN 10 represented hereafter.

00 O0 rsl

EXAMPLES

00 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, Unite de Recherches Laitieres et G6netique C- 10 Appliqu6e, INRA, Jouy en Josas. They are cultured in MRS 00 0 medium (from Man et al., J. Appl. Bacteriol., 23: 130- C-q 135, 1960) and incubated at 30 0 C, 370C or 42 0 C according to the species. The Escherichia coli TG1 strain, provided by Stratagene, is cultured in LB (Luria broth base Agar-agar 16g/L) under agitation and at 37 0

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 370C, the clarification of the preparation is obtained by adding 60 ul 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 00 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 Sphase. 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 CI incubation of one hour at -20°C, a centrifugation is carried out for 20 minutes at 15,000 g and at 4 0 C. The S10 isopropanol is eliminated and replaced by 500 pl of 00 Sethanol. A final centrifugation of 10 minutes at 15,000 g C 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 p1 of sterile water containing 10 pl of RNase at 10 pg/pl. After 15 minutes of incubation at 370C to agitate the enzyme degrading the RNAs, 10 pi 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 pM 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°C 3' ntd2 5'-ACG GCA CCT TCG TAG AAG- 18 bases Tm= 560C 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, Clal, EcoRI, HindIII, Hpal, NcoI, NotI, PstI, XbaI, XhoI (Bio-Lab). Digestion is carried out in a final volume of 40 pl containing 70 U of enzyme, 4 pl of 10X NEB buffer (Bio-Lab) and 4 to 8 pg of DNA. The incubation is carried out for 1 hour minutes at 370C.

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 O internal fragment of the ntd gene of Lactobacillus 1 helveticus amplified by PCR. The probe is purified using t the Wizard kit (Promega) in order to eliminate the PCR primers. The necessary concentration of the probe is ng/l. The DNA is marked using the ECL marking kit (Amersham). To do this, the DNA is denatured by heating C- for 5 minutes at 100°C and immediately recooled in ice. A c-I volume of marking reagent (peroxidase) then a volume of CN 10 glutaraldehyde solution are added. This solution is 00 0 incubated for 10 minutes at 37°C for covalently binding C1 the peroxidase to the DNA.

Hybridization and development. After a prehybridization of one hour at 42 0 C 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 42°C 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 NaCl 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 0 creates free ends. The digestion mixing is carried out in Sa volume of 30 pl, containing 4 pl of DNA. The DNA CI fragments amplified to be cloned should have their 5' and t 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

C of the T4 phage (Bio-Lab) and 6 units of polymerase I DNA (or Klenow fragment) (Bio-Lab), not having a 5' 3' Cl 10 exonucleic activity. The polymerization is carried out 00 Sfor 20 minutes at 11°C then the enzymes are deactivated Ci after 10 minutes at 750C. 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 pl 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 p1 containing 60 ng of insert, 26 ng of restricted pBS-SK+ plasmid, 2 units of ligase (T4 DNA ligase, Boehringer- Mannheim), overnight at 160C. 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 electrocompetent cells of the TG1 00 0 strain of E. coli. Starting from 5 ml of an overnight Ci culture at 37 0 C under agitation, 500 ml of LB medium are t inoculated. The culture is placed under agitation at 37 0

C

until an O.D.60oo 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 (C taken up in 400 ml of cold water. This preparation is cold-centrifuged for 10 minutes and at 5,000 rpm. The CI 10 pellet obtained is taken up again in 250 ml of cold 00 Swater. Following a centrifugation of 10 minutes, the Cq 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 MgCl 2 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, lug/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 OO ampicillin (100 pg/ml) are harvested by centrifugation at 15,000 for 10 minutes at 4 0 C. They are resuspended in 100 pl of a 50 mM saccharose solution, 25 mM Tris-HC1, pH 8, mM EDTA, pH 8. The alkaline lysis and the denaturation of the DNA is carried out by addition of 200 pl 0.2N NaOH, 1% SDS. The reaction medium is left for 1 minute at ambient temperature after having added 200 pl of Ci chloroform. Then 150 pl of a solution of 5M potassium acetate, glacial acetic acid are added. The reaction C 10 medium is centrifuged for 15 minutes at 13,000 g at 4°C.

00 SThe aqueous phase containing the DNA is precipitated in CI 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 pl 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 (1 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 pl of ligation buffer are 0 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 c ligated DNA is then precipitated with 100 pi of 3M sodium t acetate, pH 4.8, 700 pl of isopropanol and 2 pl of glycogen, then centrifuged for 30 minutes at 13,000 g at 4C. The pellet is rinsed in 300 pl of 70% ethanol and centrifuged for 10 minutes at 13,000 g at 4°C. The pellet c-I Cl is taken up in 25 pl of ultrapure water.

00 Sprimers. The polymerase chain reactions are carried out c-I 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, MgCl 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: 94°C 3 minutes 94°C 30 seconds to 600C (according to the 25 cycles Tm of the primers used) 1 minute 720C 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 00 reaction volume of 20 pi containing 30 ng of DNA, 4 pl of (C DyeT Mix (Perkin Elmer Biosystems) and 0.1 mM of primer.

Cycle: 96°C 1 minute Ce 960C 10 seconds 500C 5 seconds 25 cycles C' 10 600C 4 minutes OC 00 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 O phosphoribosyltransferase encoded by the gpt gene), and 0 also an activity allowing the base G to be released from the dR-G deoxynucleoside (purine nucleoside phosphorylase Sencoded 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 Sselected if an N-deoxyribosyltransferase activity is expressed in the PAK6 strain in order to carry out the exchange: dR-G A dR-A G CO 00 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 (Alul) of L. helveticus CNRZ 32 were ligated in a ColE1 plasmid (of pUC type digested by SmaI 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 00 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 t gene called ptd deviates from that of the ntd genes making them impossible to hybridize identity).

Incidentally the ntd gene of L. helveticus C controlling a Class II N-deoxyribosyltransferase activity could be isolated once again among the transforming CI 10 clones selected.

00 Cl 4. FUNCTIONAL CLONING OF THE NTD GENE OF L.

FERMENTUM.

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 0 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 (Huck, 1997) an internal fragment on the C-K ntd gene of L. helveticus was amplified. Starting from

C-I

this fragment, oligonucleotides were synthesized so as to Cl 10 obtain all the gene by inverse PCR.

00 Starting from the two ntd sequences of L.

Cl 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 Nr 7 00 deoxyribosyltransferase 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 (1 enzyme of lactobacillus helveticus and the Ndeoxyribosyltransferase I (ptd) activity with the pLH4 (1 10 plasmid containing the polynucleotide of SEQ ID No. 3 and 00coding for the ptd enzyme of lactobacillus helveticus.

Scoding for the ptd enzyme of lactobacillus helveticus.

rsl 00 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 vi vo vitro A G dG dG +A dC +T G A dC +A PAK 6- 00 PSU19 ntd 6PAK- Li ntd 6PAK- Lh ptd 6PAK- Lh ntd 6PAK- growth absence of growth PAK 6: MG1655 AguaBA::Apra, Adeo ntd Li: gene coding for the N-deoxyribosyltransf erase of Lactobacillus leichinannii ntd Lh: gene coding for the N-deoxyribosyltransf erase of Lactobacillus helveticus ntcl Lf: gene coding for the N-deoxyribosyltransf erase of Lactobacillus fermentum ptd Lh: gene coding for the purine deoxyribosyitransferase of Lactobacillus helveticus A: adenine; G: guanine; T: thymine; dG: deoxyguanosine; dO: deoxycytidine 0

REFERENCES

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

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

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

(N

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

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

CN 10 Duck et al. (1990), Biotechniques, 9, 142.

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

C( 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.

Kohler 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 1'Institut Pasteur Actualit6s 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.

Smith et Waterman (1981) Ad. App. Math. 2 482 00 Temin, (1986) Retrovirus vectors for gene transfer. In C\l Kucherlapati ed. Gene Transfer.

t 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.

00 rsl

Claims (20)

1. Purified or isolated polynucleotide of Lactobacillus having at least one N- deoxyribosyltransferase activity with an amino acid sequence chosen from the sequences, SEQ ID No.4, SEQ ID No.6, SEQ ID No.8, SEQ ID No.10, SEQ ID No.12.

2. Polypeptide isolated according to claim 1 S10 characterized in that the polypeptide of SEQ ID No.4 is 00 coded by the N-deoxyribosyltransferase coded by the ptd Lh gene of Lactobacillus helveticus.

3. Polypeptide isolated according to claim 1 characterized in that the polypeptide of SEQ ID No.6 is coded by the N-deoxyribosyltransferase coded by the ntd Lf gene of Lactobacillus fermentum.

4. Polypeptide isolated according to claim 1 characterized in that the polypeptide of SEQ ID No.8 is coded by the N-deoxyribosyltransferase coded by the ntd gene of Lactobacillus crispatus. Polypeptide isolated according to claim 1 characterized in that the polypeptide of SEQ ID No.10 is coded by the N-deoxyribosyltransferase coded by the ntd gene of Lactobacillus amylovorus.

6. Polypeptide isolated according to claim 1 characterized in that the polypeptide of SEQ ID No.12 is coded by the N-deoxyribosyltransferase coded by the ntd gene of Lactobacillus acidophilus. 00 7. Isolated polypeptide characterized in that it 0 comprises a polypeptide chosen from: a polypeptide of sequence SEQ ID No.4, SEQ ID SNo.6, SEQ ID No.8, SEQ ID No.10, SEQ ID No.12. a polypeptide variant of a polypeptide with an amino acid sequence defined in a); c) a polypeptide homologous to the polypeptide C( defined in a) or b) and comprising at least 80% identity with said polypeptide of a); S10 d) a fragment of at least 15 consecutive amino acids 00 Sof a polypeptide defined in a); a biologically active fragment of a polypeptide defined in b) or c). 8) Polypeptide according to claims 1 to 7 characterized in that it makes it possible to satisfy the guanine requirement of the PAK6 strain deposited at the CNCM on 2nd May 2001 under No. 1-2664. 9) Purified or isolated polynucleotide characterized in that it codes for a polypeptide according to claims 1 to 8. Polynucleotide according to claim 9 of sequence SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID No.11, SEQ ID No.13. 11) Isolated polynucleotide characterized in that it comprises a polynucleotide chosen from: 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 of the sequence SEQ ID No.3, SEQ 00 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 Sof at least 70% after optimal alignment with a sequence defined in a) or b); the complementary sequence or the RNA sequence corresponding to a sequence as defined in b) or c).

12. Polynucleotide according to claims 9 to 11 C 10 characterized in that its expression in the PAK6 strains 00 makes it possible to satisfy the guanine requirement of said strain.

13. Use of a polynucleotide according to claim 11 as a primer for the amplification or polymerization of nucleic sequences of N-deoxyribosyltransferases.

14. Use of a polynucleotide according to claims 9 to 12 as a probe for the detection of nucleic sequences of N-deoxyribosyltransferases. Recombinant cloning and/or expression vector comprising a polynucleotide according to one of claims 9 to 12 or expressing a polypeptide according to any one of claims 1 to 8.

16. Recombinant vector called pLH4 comprising the polynucleotide SEQ ID No.3 as present in the bacterial strain deposited at the CNCM on 30th May 2001 under No.I- 2677;

17. Recombinant vector called pLF6 comprising the polynucleotide SEQ ID No.5 as present in the bacterial 00 strain deposited at the CNCM on 30th May 2001 under No.I- S2678; S18. Recombinant vector called pLA comprising the polynucleotide SEQ ID No.20 as present in the bacterial strain deposited at the CNCM on 21st June 2001 under No.I-2689;

19. Host cell, characterized in that it is S10 transformed by a vector according to claims 15 to 18. 00 Bacterium transformed by the vector pLH4 comprising the polynucleotide SEQ ID No.3, as deposited at the CNCM on 30th May 2001 under No.I-2677;

21. Bacterium transformed by the vector pLF6 comprising the polynucleotide SEQ ID No.5, as deposited at the CNCM on 30th May 2001 under No.I-2678;

22. Bacterium transformed by the vector pLA comprising the polynucleotide SEQ ID No.9, as deposited at the CNCM on 21st June 2001 under No.I-2689;

23. Metazoic, plant or animal organism, except human, characterized in that it comprises a cell according to claim 19.

24. Process for preparing a recombinant polypeptide characterized in that a host cell according to claim 19 or a bacterium according to claims 20 to 22 is cultured under conditions allowing the expression and optionally the secretion of said recombinant polypeptide and that said recombinant polypeptide is recovered. 00 25. Recombinant polypeptide capable of being 0 obtained by a process according to claim 24. (N S26. Monoclonal or polyclonal antibody and its fragments characterized in that it binds selectively a polypeptide according to one of claims 1 to 8 or (C 27. Process for in vitro or in vivo enzymatic synthesis of deoxyribonucleotides characterized in that S10 it comprises at least one reaction stage catalyzed by one 00 N-deoxyribosyltransferase according to any one of claims 1 to 8.

28. Process according to claim 27 characterized in that said N-deoxyribosyltransferase catalyzes the exchange of a first nucleobase present in a deoxyribonucleoside by a second nucleobase.

29. Process according to claim 28 characterized in that 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. Process according to claim 29 characterized in that said second nucleobase is selected from the group composed of 6-methyl purine, 2-amino-6- methylmercaptopurine, 6-dimethylaminopurine, azacytidine, 2,6-dichloropurine, 6-chloroguanine, 6- chloropurine, 6-aza-thymine, 5-fluoro-uracile, ethyl-4- carboxylate, imidazole-4-carboxamide and 1,2,4-triazole-3-carboxamide. 00 31. Process according to claim 27 characterized in 0 Sthat said first nucleobase is selected from the group composed of adenine, guanine, thymine, uracile and hypoxanthine.

32. In vivo process according to claims 27 to 31 characterized in that it moreover comprises the stage of C( introducing into the host cell the first nucleobase present in a deoxyribonucleoside. (N 00

33. In vivo process according to claims 27 to 31 characterized in that it moreover comprises the stage of introducing into the host cell the second nucleobase present in a deoxyribonucleoside.

34. In vivo process according to claims 27 to 31 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 staggered over time. SEQUENCE LISTINGS <110> Institut PASTEUR Institut National de la Recherche Agronomique INRA (120> N-desoxyribosyl transferases of Lactobacillus, corresponding nucleotidic sequences and their applications <130>' D19532 <160> 16 <170> Patentln Ver. 2.1 <210> 1 <211> 477 <212> DNA <213> Lactobacillus helveticus NTD <220> <221> CDS <222> (477) <400> 1 atg aac aag aaa aag act tta tat ttt Met Asn Lys Lys Lys Thr Leu Tyr Phe ggt Gly gcc ggt tgg ttt Ala Gly Trp Phe aat qaa Asn Giu aag caa aac Lys Gin Asn cca aca gtt Pro Thr Val aaa Lys gct tac aaa gaa Ala Tyr Lys Glu atg gca gct tta Met Ala Ala Leu aaa gaa aat Lys Giu Asn aac caa tac Asn Gin Tyr gat tta gaa aat Asp Leu Glu Asn tat gtg ccc ctt Tyr Val Pro Leu aag ggt Lys Gly att cgc att gat Ile Arg Ile Asp cat cca gaa tac His Pro Glu Tyr t tg Leu cac aac att gaa His Asn Ile Giu t gg Trp gct tct gca acc Ala Ser Ala Thr tac Tyr 70 cac aat gat tta His Asn Asp Leu gga att aag act Gly Ile Lys Thr t ct S er 192 240 288 gat gtc atg ctt Asp Vai Met Leu qgC Gly gta tat ttg cca VTal Tyr Leu Pro gaa gaa gac gtc Glu Glu Asp VIai ggc tta Gly Leu ggc atg gaa Gly Met Glu gtt atc cca Val Ile Pro 115 ggc gtt tgt Gly Val Cys 130 ggc tac gca tta Gly Tyr Ala Leu tct Ser 105 caa gqa aaa tat Gin Gly Lys Tyr att tta ttg Ile Leu Leu 110 atg agc tgg Met Ser Trp gat gaa gat tac Asp Glu Asp Tyr aag cca atc aac Lys Pro Ile Asn gac aat gcc Asp Asn Ala atc Ile 135 aag atc agt yaa tta aaa qac ttc qac Lys Ile Ser Gin Len Lys Asp Phe Asp 2 ttt aac aag cct cgc tac aat ttc tac gac gga gct gta tat taa Phe Asn Lys Pro Arg Tyr Asn Phe Tyr Asp Giy Ala Val Tyr 145 150 155 <210> 2 <211> 158 <212> PRT <213> Lactobacillus heiveticus NTD <400> 2 Met Asn Lys Lys Lys Thr Leu Tyr Phe 1 Lys Gin Pro Thr Lys Gly Trp Ala Asp Val Gly Met Vai Ile Giy Val 130 Asn Lys Val Asp Ile Arg Ser Ala Met Leu Giu Leu 100 Pro Asp 115 Cys Asp 5 Al a Le u Ile Thr Gly Gly Giu As n Tyr Giu Asp Tyr 70 Val Tyr Asp Al a Lys Asn Glu 55 His Tyr Al a Tyr Ile 135 Giu Ser 40 His As n Leu Leu Gi y 120 Lys Ala 25 Tyr Pro Asp Pro Ser 105 Lys Ile Gly Met Val1 Glu Leu Glu 90 Gin Pro Ser Al a Al a Pro Tyr Val1 75 Giu Gly Ile Glu Gly Al a Leu Leu Gly Giu Lys As n Leu Trp Leu Glu His Ile Asp Tyr Leu 125 Lys Phe Lys As n As n Lys Val1 Ile 110 Met Asp Asn Glu Gin Ile Thr Gly Leu Ser Phe Giu Asn T yr Glu Ser Leu Leu Trp Asp 140 Phe 145 Asn Lys Pro Arg Asn Phe Tyr Asp Gly 155 Ala Vai Tyr <c210> 3 <211> 504 <212> DNA <213> Lactobacillus helveticus PTD <220> <221> GUS <222> (504) <400> 3 atg aaa gca gta gtt cca aca gga aaa att tat tta ggc tca cca ttt Met Lys Ala Val Val Pro Thr Gly Lys Ile Tyr Leu Gly Ser Pro Phe 1 5 10 tac agc gat qct caa aga gaa aga gca gct aag gca aaa gag ttg tta Tyr Ser Asp Ala Gin Arg Giu Arg Ala Ala Lys Ala Lys Giu Leu Leu 25 gca aaa aat cta agc atc gcg cac gtc Ala Lys Asn Leu Ser Ile Ala His Val 40 ttc Phe atg Met aa t As n ggc Gly atc Ile ct g Leu acc Thr gt t Val1 gcc Ala tct Ser ttg Leu atg Met gat Asp t gg Trp act Thr gcc Ala qtg Val 115 atc Ile cct Pro cgg Arg t gt Cys ttt Phe 100 cca Pro gca Al a ga t Asp ga t Asp ggc Gly gaa Glu ttc Phe caa Gin gaa Giu gca Ala 70 qtc Val att Ile act Thr ggc Gly aac Lys act Thr ttc Phe ggc Gly gag Giu gta Val aat Asn tac Tyr tta Leu t tc Phe cat His 120 acc Thr cct Pro caa Gin tat Tyr at g Met 105 ccc Pro acc I'hr 3 -ttc *Phe gaa Giu aat As n ga t Asp 90 cgt Arg gaa Giu atc Ile ttC ccc ttt gat gat Phe Pro Phe Asp Asp att I le gat Asp 75 atg Met gcg Al a aaa Lys att Ile ggc Gly t ta Leu ga t Asp atg Met gaa Glu gat Asp 140 ggc Gly act Thr caa Gin cat His aag Lys 125 9gc Gly atc Ile ggt Giy tt a Le u aag Lys 110 a aa. Lys aat Asn aga Arg att. Ile ga t Asp cog Pro atg Met act Thr 9gt Gly ago: Ser tog Ser gao Asp gtg Val1 a ac Asn gaa Glu 144 192 240 288 336 384 432 130 135 ttt Phe 145 qt t Val gaa Glu ogo Arg ota Leu tao T yr got Ala ggt Gi y 165 gat Asp 150 ato Ile aac tto aac gaa Asn Phe Asn Glu 155 taa tgt cot ttt aat Cys Pro Phe Asn <210> 4 <211> 167 <212> PRT <213> Lactobacillus <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 helveticus PTD Pro Thr Gly Lys Arg Giu Arg Ala 25 Ile Ala His Val 40 Giu Lys Asn Pro 55 Ala Thr Tyr Gin 70 Val Phe Leu Tyr Ile Tyr 10 Ala Lys Phe Phe Giu Ile Asn Asp 75 Asp Met 90 Leu Gly Ala Lys Pro Phe Gly Gly Leu Thr Asp Gin Ser Glu Asp Ile Gl y Le u Pro Leu Asp Arcj Ile Asp Phe Leu Gly Se r Ser Asp Gly Ile Leu Phe 145 Val1 Al a Val1 115 Ile Lys Gly Phe 100 Pro Al a Leu Tyr Glu Phe Gin Ala Gly 165 Ile Thr Gly Asp 150 Ile Gly Phe Met Arg Ala Met His Lys Pro Val 105 110 Giu His Pro Glu Lys Glu Lys Lys Met Asn 120 125 Val Thr Thr Ile Ile Asp Giy Asn Thr Glu 135 140 Tyr Asn Phe Asn Giu Cys Pro Phe Asri Pro 155 160 Tyr <210> <211> 516 <212> DNA <213> Lactobacillus fermentum NTD <220> <221> CDS <222> <400> ttg aaa aat Leu Lys Asn 1 agc ttc ttc Ser Phe Phe caa cta gaa Gin Leu Giu ttc caa tat Phe Gin Tyr ggc aqc ctc Gly Ser Leu gta gga act Val Giy Thr gac qaa gga Asp Glu Gly cct atc gtt Pro Ile Val 115 (504) acc Thr cca gtt gct aac act aaa att tac ctg gct acc: Pro Vai Ala Asn Thr Lys Ile Tyr Leu Ala Thr 10 aac gaa gaa caa cgt Asn Glu Giu Gin Arg qcc aac ccg act gtc Ala Asn Pro Thr Val 40 aaa gat gca cgc qta Lys Asp Ala Arg Val 55 gaa tgg caa att gcc Glu Trp Gin Ile Ala 70 tcc gat gtc tgc gtt Ser Asp Val Cys Val att tgt atg gaa atc Ile Cys [let Giu Ile 100 tta Cta cct ttt act Leu Leu Pro Phe Thr 120 gcc cgc atc cct caa gCt Cta gcc Al a 25 ggC Cly ga c Asp act Thr gc t Aila 9gc Gi y 105 aag Lys Arg gtt ValI tc Ser tac Tyr tta Leu 90 atg H'e t aaa Lys Ile Pro Gin Ala Leu Ala gtt cac cag cca ttc gat Val His Gin Pro Phe Asp gat cct gcc ggc gtc ttt Asp Pro Ala Gly Val Phe aat aac gac ctc aac gcg Asn Asn Asp Leu Asn Ala 75 tac gat atg gac caa att Tyr Asp Met Asp Gin Ile ttc gtc gcc ctc cat aaa Phe Val Aia Leu His Lys 110 gat aag tct gct tat gaa Asp Lys Ser Ala Tyr Glu 125 48 96 144 192 240 288 336 384 gct aac cta atq cta gca cgg ggt Ala Asn Leu Met Leu Ala Arq Gly gt a Val1 act acc tgg ttg gaa cct aat Thr Thr Trp Leu Giu Pro Asn 130 gao ttt Asp Phe 145 Cct ttc Pro Phe agt coo tta Ser Pro Leu cca cca tto Pro Pro Phe 165 135 140 aaa gac ttt aac ttt aac cac oca atg got oaa 480 Lys Asp Phe Asn Phe Asn His Pro Met Ala Gin 150 155 160 aag gtt ttc taaotaacct aa 516 Lys Val Phe <210> 6 <211> 168 <212> PRT <213> Lacto <400> 6 Leu Lys Asn 1 Ser Phe Phe Gin Leu Giu Ph e Gly Val1 Asp Pro Ala Asp 145 Pro Gin Ser Gly Gi u Ile Asn 130 Phe Phe Tyr Leu Thr Gly Val 115 Le u Ser Pro bacillus fermentum NTD Thr Asp Pro Val Ala Ai 5 Asn Giu Glu Gin Arg Al 202 Ala Asn Pro Thr Val Gi 40 Lys Asp Ala Arg Val As 55 Glu Trp Gin Ile Ala Th 70 Ser Asp Val Cys Val Al Ile Cys Met Glu Ile Gl 100 10~ Leu Leu Pro Phe Thr Ly~ r a 5 iThi Arc Val Ser Tyr Leu 90 Met Lys Thr Phe Lys Ile Val Asp As n 75 Tyr Phe Asp Thr Asn 155 Ile Pro His Pro As n Asp Val1 Lys rrp 140 iis Tyr Gin Gln Al a Asp Met Al a Ser *Leu Ala Pro Gly Leu Asp Leu 110 Ala Ala Leu Phie ValI Asn Gin Hiis T'yr Thr Ala Asp Phe Ala Ile Lys Glu Met Pro Pro Leu Leu Phe 165 Ala Lys 150 Lys Arg 135 Asp Val1 Va As r 125 Leu Pro Glu Met Asn Gin 160 <210> 7 <211> 255 <212> DNA <213> Lactobacillus orispatns NTD <220> <221> GOS <222> (254) <400> 7 ac aac cag tac aag ggt atc cgc gtt gat gaa cac Oct gaa tac ttg Asn Gin Tyr Lys Gly Ile Arg Val Asp Giu His Pro Glu Tyr Leu 1 5 10 cac gac att gaa tgg gca tca got aco tao cat aac gac tta gta ggg His Asp Ile Glu Trp Ala Ser Ala Thr Tyr His Asn Asp Leu Val Gly 25 att aag tcc agc gac atc atg ctt ggc gtt tao ttg cct gaa gaa gaa Ile Lys Ser Ser Asp Ile Met Leu Gly Val Tyr Leu Pro Giu Giu Giu 40 gat qtt ggt ctg gga atg gaa ott ggc tat gco Ott tca aaa gqc aag Asp Val Gly Leu Gly Met Giu Leu Gly Tyr Ala Leu Ser Lys Gly Lys 55 tao atc ttg ttg gta att cot gat gaa gat tao ggt aag oca ato aac Tyr Ile Leu Leu Val Ile Pro Asp Giu Asp Tyr Gly Lys Pro Ile Asn 70 tta atg ago tgg ggo a Leu Met Ser Trp Gly 47 143 191 239 255 <210> 8 (211> 84 <212> PRT <213> Lactobaoiilus <400> 8 Asn Gin Tyr Lys Giy 1 5 Asp Ile Giu Trp Ala Lys Ser Ser Asp Ile Val Gly Leu Gly Met Ile Leu Leu Vai Ile Met Ser Trp Gly orispatus NTD Ile Arg Val Asp Ser Aia Thr Tyr 25 Met Leu Gly Val 40 Giu Leu Gly Tyr 55 Pro Asp Giu Asp 70 Giu 10 His Tyr Ala Tyr His Asn Le u Leu Gly 75 Pro Asp Pro Ser Lys Giu Le u Gi u Lys Pro Tyr Val1 Giu Gly Ile Leu Gly Glu Lys Asn His Ile Asp Tyr Leu <210> 9 <211> 399 <212> DNA <213> Lactobacillus amylovorus NTD <220> <221> CDS <222> <400> 9 atg gaa Met Glu 1 got tta aaq aag aac cct act gtt gac tta gaa aac agt tac Ala Leu Lys Lys Asn Pro Thr Val Asp Leu Giu Asn Ser Tyr 10 gto Val gaa Glu tta Leu gaa Glu oaa Gin oca Pro cca Pro tat Tyr gtt Val gaa Glu ggt Gly atc Ile ctt Leu tta Leu ggt Gly gaa Glu aaa Lys aac Asn gat Asp cac His att Ile gat Asp tac Tyr ttg Leu 100 aac Asn gao Asp aag Lys gtt Val atc Ile atg Met caa Gin att Ile tot Ser ggc Gly 70 ttg Leu ago Ser tao Tyr gaa Glu toa Ser 55 ott Leu ott Leu tgg Trp aaa Lys tgg Trp 40 gao Asp ggg Gly gto Val ggc Gly ggo Gly 25 gca Ala gta Val atg Met ato Ile gtt Va1 105 ato lie toa Ser atg Met gaa Glu coct Pro 90 tgo Cys cgo Arg tot Ser otc Leu ott Leu 75 gao Asp gao Asp gtt Val aco Thr ggt Gly ggc Gly gaa Glu aao Asn gat Asp tao Tyr gtt Val tao Tyr gao Asp gta Val gaa Glu cac His tat Tyr gca Ala tat Tyr ato Ile 110 oac His aat Asn tta Leu ttg Leu ggt Gly aag Lys oca Pro gao Asp cct Pro tot Ser aag Lys ato Ile 48 96 144 192 240 288 336 ago gaa ttg aaa gao tto gao ttt aao Ser Giu Leu Lys Asp Phe Asp Phe Asn 115 120 gat ggt got gto tat Asp Gly Ala Val Tyr 130 <210> <211> 133 <212> PRT <213> Laotobacillus amylovorus NTD <400> Met Giu Ala Leu Lys Lys Asn Pro Thr 1 5 Vai Pro Leu Asp Asn Gin Tyr Lys Gly 25 Glu Tyr Leu His Asp Ile Glu Trp Ala 40 Leu Val Gly Ile Lys Ser Ser Asp Val I 55 Glu Glu Giu Asp Val Gly Leu Gly Met 70 Gin Gly Lys Tyr Ile Leu Leu Val Ile aga oct cgo Arg Pro Arg Val 10 Ile Ser Met ilu Pro 90 Asp Arg Ser Leu Leu 75 Asp Leu Vai Thr Gly Gly Glu tto Phe 125 Glu Asp Tyr Val Tyr I Asp T 399 aac ttt tao Asn Phe Tyr Asn Glu His yr la 'yr Ser Tyr His Pro Asn Asp Leu Pro Leu Ser Gly Lys Pro Ile Asn Leu Met Ser Trp Gly Val 100 105 Ser Giu Leu Lys Asp Phe Asp Phe Asn 115 120 Cys Asp Asn Arg Pro Arg Val Ile Lys Ile 110 Phe Asn Phe Tyr 125 Asp Gly Ala Val Tyr 130 <210> 11 <211> 480 <212> DNA <213> Lactobacilius acidophilus NTD <220> <221> COS <222> <400> 11 atg atg gca Met Met Ala I gaa aag caa. Giu Lys Gin aac cct act Asn Pro Thr aaa. aca Lys Thr 5 aaa act tta, tat Lys Thr Leu Tyr ggc gct ggt tgg Gly Ala Gly Trp ttt aat Phe Asn aat As n aag qct tat aaa. Lys Ala Tyr Lys gct atg qaa gct Ala Met Giu Ala tta aaa. caa Leu Lys Gin gaa aat caa. Glu Asn Gin qtt gat ttg gaa Val Asp Leu Giu agt tat gtt cca Ser Tyr Val Pro ctt Leu tat aaa Tyr Lys gat att cgt gtt Asp Ile Arg Val gat Asp 55 gaa cat cct gaa Glu His Pro Glu t ac Tyr tta cac gac att Leu His Asp Ile ga a Gi u tgg gca tct gct Trp Ala Ser Ala tat cac aac gac Tyr His Asn Asp att ggt atc aaa Ile Giy Ile Lys tct Ser 192 240 288 tca gat att atg Ser Asp Ile Met tta Le u ggg gtt tac tta Giy Val Tyr Leu gaa gaa gaa gat Giu Giu Giu Asp gtt ggt Val Giy ctt ggt atg Leu Gly Met ctc qtt att Leu Val Ile 115 gaa Glu 100 ctt ggc tac gca Leu Gly Tyr Ala tca caa ggc aaa Ser Gin Gly Lys tat atc tta Tyr Ile Leu 110 ttg atg agt Leu fNet Ser 336 384 cct gac gaa gat Pro Asp Giu Asp ggc aag cct atc Gly Lys Pro Ile tgg ggt Trp Gly 130 gta tgt gat aac Val Cys Asp Asn att aaq atc aqc Ile Lys Ile Ser ttg aag gac ttc Leu Lys Asp Phe 432 gac ttc aat aag cca CgC ttt aac ttc tat gat ggc gct gta tat taa Asp Phe Asn Lys Pro Arg Phe Asn Phe Tyr Asp Gly Ala Vai Tyr 145 150 q <210> 12 (211> 159 <212> PRT <213> Lactobacillus acidophilus NTD (400> 12 Met Met Ala 1 Glu Lys Asn Pro Tyr Lys Glu Trp Ser Asp Leu Gly Leu Val Gin Thr Asp Al a Ile Met Ile Lys Asn Val Ile Ser Met Glu 100 Pro *Thr 5 Lys Asp Arg Ala Leu Leu Asp Lys Thr Leu Tyr Phe Gly Ala Gly Trp Phe Asn 10 Ala Tyr Leu Glu Val Asp 55 Thr Tyr 70 Gly Val Gly Tyr Glu Asp Lys As n 40 Glu His Tyr Al a Tyr 120 Ala 25 Ser His As n Leu Le u 105 Gly Ala. Tyr Pro Asp Pro 90 Ser Lys Met Val1 Giu Leu 75 Glu Gin Pro Glu Pro Tyr Ile Glu Gly Ile Al a Leu Leu Gl y Glu Lys Asn 125 Leu Glu His Ile Asp Tyr 110 Leu Lys As n Asp Lys ValI Ile Met Gin Gin Ile Ser Gly Leu Ser Trp Gly Val Cys Asp Asn Ala 130 135 Ile Lys Ile Ser Glu Leu Lys Asp Phe 140 Asp 145 Phe Asn Lys Pro Arg 150 Phe Asn Phe Tyr Asp 155 Gly Ala Val Tyr <210> 13 <211> 795 <212> DNA (213> Lactobacillus heiveticus NTD <220> <221> CDS <222> (140)..(616) <220> <223> n signifies any nucleotide g, c or t/u <400> 13 aaaaaaattt tcagtattag tcattgaatt ttaccttcca ttatgqaatt actattttta qcgtaagtta acaagacqtt tttttcaatc gaaaatatgt taaagttaat tcgtcagcaa 120 tttttatggg ganaaaatt atg aac aag aaa aag act tta tat ttt ggt gcc 172 Met Asn Lys Lys Lys Thr Leu Tyr Phe Gly Ala 1 5 00 00 ggt tgg ttt aat gaa aag caa aac Gly Trp Phe Asn Giu Lys Gin Asn gct tac aaa gaa gca atq gca Ala Tyr Lys Giu Ala Met Ala 220 get Ala ctt tta Le u gaa aaa Lys aac gaa Giu caa aat Asn tac eca Pro aag aca gtt gat tta gaa Leu Glu Asn Gin Tyr Lys ttg Leu gga Gi y gaa Gi u aaa Lys aac Asn tta cac His att Ile gac Asp tat Tyr tta Leu 125 aaa aac Asn aaq Lys gte Vali at t Ile 110 atg Met gac at t Ile act Thr ggc Gly tta Leu agc Ser ttc gaa Glu tct Ser tta Leu ttg Leu t gg Trp gac tqq Trp 65 gat Asp ggc Gly gt t Val1 ggc Gly ttt Thr Val 35 ggt att Gly Ile get tct Ala Ser gtc atg Vai Met atg gaa Met Giu ate cea Ile Pro 115 Asp ege Arg gca Al a ct t Leu ctg Leu 100 gat Asp Le. at t Ile aec Thr ggC Gly 85 ggc Giy gaa Gi u iGiu gat Asp tac Tyr 70 qta. Val1 tae Tyr gat Asp gee Ala t ae Tyr 150 aat agt tat Asn Ser Tyr gaa eat eea Giu His Pro eac aat gat His Asn Asp tat ttg eca Tyr Leu Pro gea tta tet Ala Leu Ser 105 tac ggc aag Tyr Gly Lys 120 ate aag ate Ile Lys Ile 135 aat tte tae Asn Phe Tyr I1 gtg Val gaa Giu tta Leu gaa Giu eaa Gin cca Pro agt Ser ;a e .sp ec Pro tae Tyr gta ValI gaa. Glu gga Gly ate Ile gaa Giu qga. Gly 155 268 316 364 412 460 508 556 604 gtt Vali 130 aae tgt gae aat Asp eet Pro Asn ege Arg Leu Lys Asp Phe Asp Phe Asn gct gta tat taa aaaataagca aaetaaatga ectategett aaaaattgcg Ala Vai Tyr ataggteatt ttttaatatt atetgteatg tataaaatet ttettaataa atatacteca agtgattttc caaaaaaatt attattetat aeccaettca tatggaagte egagtcactt atgtaaatea tatatcact <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 Glu 1 5 10 Lys Gin Asn Lys Ala Tyr Lys Giu Ala Met Ala Ala Leu Lys Giu Asn 25 Pro Thr Val Asp Leu Giu Asn Ser Tyr Vai Pro Leu Giu Asn Gin Tyr 40 656 716 776 795 00 Lys Gly Ile Arg Ile Asp Glu His Pro Glu Tyr Leu His Asn Ile Glu C150 55 Trp Ala Ser Ala Thr Tyr His Asn Asp Leu Val Cly 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 Giu Leu Gly Tyr Ala Leu Ser Gin Gly Lys Tyr Ile Leu Leu C1100 105 110 __Val Ile Pro Asp Glu Asp Tyr Gly Lys Pro Ile Asn Leu Met Ser Trp 115 120 125 00Gly 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> <211> 18 <212> DNA (213> Lactobacillus ieichmannii NTD1 (400> agacgatcta cttcggtg 18 <210> 16 <211> 18 (212> DNA <213> Lactobacillus leichmannii NTD2 <400> 16 acggcacctt cgtagaag