Imported: 10 Mar '17 | Published: 09 Oct '08
USPTO - Utility Patents
The invention concerns novel polypeptides and their fragments, isolated from Lactobacillus, having at least a N-deoxyribosyl transferase activity, the polynucleotides encoding said polypeptides, cloning and/or expression vectors including said polynucleotides, cells transformed by said vectors and specific antibodies directed against said polypeptides. The invention also concerns a method for enzymatic synthesis of deoxyribonucleosides.
The present invention relates to the field of biology, and more particularly to the microbiological production of base analogues. The present invention relates to new polypeptides and their fragments, isolated from Lactobacillus, having at least one 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 [Prigaud et al., 1992]. Moreover the insecticidal and herbicidal properties of certain nucleoside antibiotics make them potential agents in the sector of agrichemicals and the environment [Isono, 1988]. The industry uses two methods for producing these analogues, organic synthesis and biocatalytic conversion (enzymatic conversion and microbiological conversion), which have advantages and, conversely, drawbacks. Organic synthesis makes it possible to achieve extremely widely varied chemical structures but requires multiple stages and is expensive in terms of reagents and solvents. On the other hand, the biocatalytic processes allow easy production in an aqueous medium but limited to a small number of possible compounds due to the specificity of the enzymes, which allow a limited range of analogues in the place of their physiological substrates. The phosphorylase nucleosides and N-deoxyribosyltransferase, which result from the purine and pyrimidine salvage pathways in the bacteria, are the enzymes most used for these enzymatic conversions (Krenisky et al., 1981).
There is therefore an urgent requirement to obtain enzymes for conversion of nucleosides and their derivatives, having a broadened enzyme activity in order to diversify the industrial production of these compounds. This is the technical problem which the inventors of the present invention propose to resolve.
The N-deoxyribosyltransferase of Lactobacillus leichmannii as well as that of L. helveticus, partially purified or not purified, is shown to be the best glycosyl group donor and tolerates a considerable number of structural variations on the base. This enzyme has been used for producing a certain number of analogues among which should be cited 2-chloro,2-deoxyadenoside (Carson et al., 1984), 2,3-dideoxynucleosides of natural bases (Carson and Wasson, 1988), or several pyrazolo(3,4-d)pyrimidine and triazolo(4,5-d)pyrimidine 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 N-deoxyribosyltransferase activity of different strains of lactobacilli. This variety of N-deoxyribosyltransferase enzymes makes it possible to increase the chances of obtaining enzymes with specificity broadened by mutations in the wild-type genes or by chimeras of these wild-type genes.
Two classes of N-deoxyribosyltransferase have been distinguished (Danzin and Cardinaud, 1976), the first (Class I) designated ptd (for purine transdeoxyribosylase) catalyzing exclusively the exchange of deoxyribose between two purines:
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:
Only two genes specifying Class II enzymes, designated ntd, have been reported to date (Hck, 1997; dbj|BAA92683.2|(ABO39914)).
A subject of the present invention is therefore an isolated or purified polypeptide of Lactobacillus having at least one N-deoxyribosyltransferase 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-deoxyribosyltransferase of SEQ ID No.6 coded by the ntd Lf gene of Lactobacillus fermentum.
According to a fourth embodiment, the polypeptide according to the invention is the N-deoxyribosyltransferase of SEQ ID No.8 coded by the ntd gene of Lactobacillus crispatus.
According to a fifth embodiment, the polypeptide according to the invention is the N-deoxyribosyltransferase of SEQ ID No.10 coded by the ntd gene of Lactobacillus amylovorus.
According to a sixth embodiment, the polypeptide according to the invention is the N-deoxyribosyltransferase 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) a polypeptide of sequence SEQ ID No.2, SEQ ID No.4, SEQ ID No.6, SEQ ID No.8, SEQ ID No.10, SEQ ID No.12, SEQ ID No.14; (b) a polypeptide variant of a polypeptide with amino acid sequences defined in a); (c) a polypeptide homologous to the polypeptide defined in (a) or (b) and comprising at least 80% identity, preferably 85%, 87%, 90%, 95%, 97%, 98%, 99% identity with said polypeptide of a); (d) a fragment of at least 15 consecutive amino acids, preferably 17, 20, 23, 25, 30, 40, 50, 100, 250 consecutive amino acids of a polypeptide defined in a), b) or c); and (e) a biologically active fragment of a polypeptide defined in a), b) and c).
The polypeptide according to the invention is characterized in that it makes it possible to satisfy the guanine requirement of certain bacterial strains such as PAK6 which is a strain of E. coli the two genes of the guaBA operon of which, which control the conversion of IMP to XMP then to GMP, as well as those of the deoCABD operon which control the degradation of the deoxynucleosides, have been deleted. In fact, these strains, in order to survive or grow, require a supply of deoxyguanosine (dR-G) to the culture medium and the presence of a N-deoxyribosyltransferase activity of a polypeptide according to the invention in order to carry out the exchange: dR-G+A-dR-A+G.
In the present description, the term polypeptide will be used equally to designate a protein or a peptide.
By variant polypeptide is meant all of the mutated polypeptides which can exist naturally, in particular in the human being, and which correspond in particular to truncations, substitutions, deletions and/or additions of amino acid residues.
By homologous polypeptide is meant the polypeptides having, relative to the natural deoxyribosyltransferases of Lactobacillus according to the invention, certain modifications such as in particular a deletion, addition or substitution of at least one amino acid, a truncation, an elongation and/or chimeric fusion. Among the homologous polypeptides, those are preferred, the amino acid sequence of which has at least 80% identity, preferably at least 85%, 87%, 90%, 93%, 95%, 97%, 98%, 99% identity with the amino acid sequences of the polypeptides according to the invention. In the case of a substitution, one or more consecutive or non-consecutive amino acids can be replaced by equivalent amino acids. The expression equivalent amino acid is here intended to designate any amino acid capable of being substituted for one of the amino acids of the basic structure, without however modifying the characteristics or essential functional properties, such as their biological activities (i.e. of deoxyribosyltransferase), corresponding polypeptides such as the in vivo induction of antibodies capable of recognizing the polypeptide the amino acid sequence of which is comprised in the amino acid sequence SEQ ID No.2, SEQ ID No.4, SEQ ID No.6, SEQ ID No.8, SEQ ID No.10, SEQ ID No.12, SEQ ID No.14 or one of its fragments. These equivalent amino acids can be determined either on the basis of their homology of structure with the amino acids for which they are substituted, or on the basis of the results of tests for cross-species reactivity to which the different polypeptides are capable of giving rise. By way of example, there will be mentioned the possibilities of substitutions capable of being carried out without resulting in a more profound modification of biological activities of the corresponding modified polypeptides, the replacements, for example of leucine by valine or isoleucine, of aspartic acid by glutamic acid, of glutamine by asparagine, of arginine by lysine etc., it being naturally possible to envisage the reverse substitutions under the same conditions.
By polypeptide fragment is meant a polypeptide comprising at least 15 consecutive amino acids, preferably 17, 20, 23, 25, 30, 40, 50, 100, 250 consecutive amino acids. The polypeptide fragments according to the invention obtained by cleavage of said polypeptide by a proteolytic enzyme, by a chemical reagent, or by placing said polypeptide in a very acid environment, also form part of the invention.
By biologically active fragment is meant in particular a fragment of an amino acid sequence of a polypeptide according to the invention, having at least one of the characteristics or functional properties of the polypeptide according to the invention, in particular in that it comprises an N-deoxyribosyltransferase activity. The variant polypeptide, the homologous polypeptide or the polypeptide fragment according to the invention has at least 10%, preferably 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% of the N-deoxyribosyltransferase activity.
Different protocols known to a person skilled in the art have been described for introducing mutations into the polypeptides. Among these, there should be mentioned by way of example, the polymerase chain reaction (PCR) in the presence of manganese (Cadwell et al., 1992). The mutations can be introduced either randomlyin this case the mutagenesis stage is following by a stage of screening the mutant of interesti.e. in targeted a manner. In the latter case, the mutations are preferably introduced at the level of the catalytic site of the N-deoxyribosyltransferases 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 80%, 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 80%, preferably at least 85%, 90%, 95%, 98% and 99% after optimal alignment with a reference sequence, is meant the polypeptides having certain modifications relative to the reference polypeptide, such as in particular one or more deletions, truncations, an elongation, a chimeric fusion, and/or one or more substitutions.
Among the polypeptides the amino acid sequence of which has a percentage identity of least 80%, preferably at least 85%, 90%, 95%, 98% and 99% after optimal alignment with the sequences SEQ ID No.2, SEQ ID No.4, SEQ ID No.6, SEQ ID No.8, SEQ ID No.10, SEQ ID No.12, SEQ ID No.14 or with one of their fragments according to the invention, the variant polypeptides coded by the variant peptide sequences as previously defined are preferred, in particular the polypeptides, the amino acid sequence of which has at least one mutation corresponding in particular to a truncation, deletion, substitution and/or addition of at least one amino acid residue relative to the sequences SEQ ID No.2, SEQ ID No.4, SEQ ID No.6, SEQ ID No.8, SEQ ID No.10, SEQ ID No.12, SEQ ID No.14 or with one of their fragments; more preferably, the variant polypeptides having at least one mutation which reduces the specificity of the polypeptide according to the invention for its substrate, such that the variant polypeptides according to the invention are capable of catalyzing a larger variety of substrate, in order to obtain a wider range of base analogues.
The invention also relates to a purified or isolated polynucleotide characterized 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.1, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID No.11, SEQ ID No.13.
The isolated or purified polynucleotide according to the invention is characterized in that it comprises a polynucleotide chosen from (a) SEQ ID No.1, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID No.11, SEQ ID No.13; (b) the sequence of a fragment of at least 15 consecutive nucleotides, preferably at least 18, 21, 24, 27, 30, 35, 40, 50, 75, 100, 150, 200 consecutive nucleotides of the sequence SEQ ID 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; (c) a nucleic sequence having a percentage identity of at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, 98%, 99% after optimal alignment with a sequence defined in a) or b); (d) the complementary sequence or the RNA sequence corresponding to a sequence as defined in a), b) or c).
The polynucleotide according to the invention is also characterized in that its expression in the host cells, in particular the bacterial strains such as PAK6, make it possible to satisfy the guanine requirement of said strain. The PAK6 strain was deposited at the CNCM on 2 May 2001 under No. I-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 (guaBA::gm deo-11) is auxotrophic for guanine in minimal glucose medium.
By nucleic acid, nucleic sequence or nucleic acid sequence, polynucleotide, oligonucleotide, polynucleotide sequence, nucleotide sequence, terms which will be used interchangeably in the present description, is meant a precise chain of nucleotides, modified or unmodified, making it possible to define a fragment or a region of a nucleic acid, comprising or not comprising non-natural nucleotides, and being able to correspond equally well to a double-stranded DNA, a single-stranded DNA and transcription products of said DNAs, and/or a fragment of RNA.
It must be understood that the present invention does not relate to the nucleotide sequences in their natural chromosomal environment, i.e. in the natural state. They are sequences which have been isolated or purified, i.e. they have been collected, directly or indirectly, for example by copying, their environment having been at least partially modified. Thus the nucleic acids obtained by chemical synthesis are also meant.
By polynucleotide of complementary sequence is meant any DNA the nucleotides of which are those of SEQ ID No.1, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID No.11, SEQ ID No.13 or part of SEQ ID No.1, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID No.11, SEQ ID No.13, and the orientation of which is reversed.
By percentage identity between two nucleic acid or amino acid sequences within the meaning of the present invention, is meant a percentage of nucleotides or amino acid residues identical between the two sequences to be compared, obtained after the best alignment, this percentage being purely statistical and the differences between the two sequences being distributed at random and throughout their length. By best alignment or significant alignment is meant the alignment for which the percentage identity determined as follows is the highest. The comparisons of sequences between two nucleic acid or amino acid sequences are carried out in standard manner by comparing these sequences after having aligned them in significant manner, said comparison having been carried out by segment or by window of comparison in order to identify and compare the local regions of sequence similarity. The significant alignment of the sequences for the comparison can be carried out, apart from manually, by means of the local homology algorithm of Smith and Waterman (1981), by means of the local homology algorithm of Neddleman and Wunsch (1970), by means of the similarity search method of Pearson and Lipman (1988), by means of computer software using these algorithms (GAP, BESTFIT, BLAST P, BLAST N, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.). In order to obtain the significant alignment, the BLAST program is used, with the BLOSUM 62 matrix. The PAM or PAM250 matrices can also be used.
The percentage identity between two nucleic acid or amino acid sequences is determined by comparing these two sequences aligned in significant manner, the nucleic acid or amino acid sequence to be compared being able to comprise additions or deletions relative to the reference sequence for a significant alignment between these two sequences. The percentage identity is calculated by determining the number of identical positions for which the nucleotide or the amino acid residue is identical between the two sequences, by dividing this number of identical positions by the total number of positions compared and by multiplying the result obtained by 100 in order to obtain the percentage identity between these two sequences.
By nucleic sequences having a percentage identity of at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, 98% and 99% after significant alignment with a reference sequence, is meant the nucleic sequences having, relative to the reference nucleic sequence, certain modifications such as in particular a deletion, truncation, elongation, chimeric fusion, and/or a substitution, in particular a point substitution, and the nucleic sequence of which has at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, 98% and 99% identity after significant alignment with the reference nucleic sequence. These are preferably sequences the complementary sequences of which are capable of being hybridized specifically with the sequences SEQ ID No.1, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID No.11, SEQ ID No.13 of the invention. Preferably, the specific conditions of hybridization or of high stringency will be such that they ensure at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, 98% and 99% identity after significant alignment between one of the two sequences and the complementary sequence of the other. A hybridization under conditions of high stringency signifies that the conditions of temperature and ionic force are chosen in such a manner that they allow the maintenance of the hybridization between two fragments of complementary DNA. By way of illustration, conditions of high stringency of the hybridization stage for the purposes of defining the polynucleotide fragments described above, are advantageously the following: the DNA-DNA or DNA-RNA hybridization is carried out in two stages: (1) prehybridization at 42 C. for 3 hours in phosphate buffer (20 mM, pH 7.5) containing 5SSC (1SSC corresponds to a 0.15 M NaCl+0.015 M sodium citrate solution), 50% formamide, 7% sodium dodecyl sulphate (SDS), 10Denhardt's, 5% dextran sulphate and 1% salmon sperm DNA; (2) hybridization per se for 20 hours at a temperature depending on the size of the probe (i.e.: 42 C., for a probe of size100 nucleotides) followed by two 20-minute washings at 20 C. in 2SSC+2% SDS, one 20-minute washing at 20 C. in 0.1SSC+0.1% SDS. The last washing is carried out in 0.1SSC+0.1% SDS for 30 minutes at 60 C., for a probe of size100 nucleotides). The hybridization conditions of high stringency described above for a polynucleotide of defined size, can be adapted by a person skilled in the art for oligonucleotides of a larger or smaller size, according to the teaching of Sambrook et al., 1989.
Among the nucleic sequences having a percentage identity of at least 70%, preferably at least 75%, 80%, 85%, 90%, 95%, 98% and 99% after significant alignment with the sequence according to the invention, the variant nucleic sequences of SEQ ID No.1, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID No.11, SEQ ID No.13 or their fragments are also preferred, i.e. all of the nucleic sequences corresponding to allelic variants, i.e. individual variations 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.
More particularly, the invention relates to a purified or isolated nucleic acid according to the present invention, characterized in that it comprises or is constituted 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.1, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9, SEQ ID No.11, SEQ ID No.13.
The primers or probes, characterized in that they comprise a 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 N-deoxyribosyltransferase, by using in particular an amplification method such as the PCR method, or a related method. According to the invention, the polynucleotides which can be used as probes or primers in processes for the detection, identification, assay or amplification of the nucleic sequence, have a minimal size of 10 bases, preferably at least 15, 18, 20, 25, 30, 40, 50 bases.
According to one embodiment, the primers according to the invention are chosen from the sequences SEQ ID No.15 and SEQ ID No.16.
The polynucleotides according to the invention can thus be used as primers and/or probes in processes implementing in particular the PCR (polymerase chain reaction) technique (Rolfs et al., 1991). This technique requires the choice of pairs of oligonucleotide primers framing the fragment which has to be amplified. Reference can, for example, be made to the technique described in U.S. Pat. No. 4,683,202. The amplified fragments can be identified, for example according to agarose or polyacrylamide gel electrophoresis, or according to a chromatographic technique such as gel filtration or ion exchange chromatography, then sequenced. The specificity of the amplification can be controlled by using as primers the nucleotide sequences of polynucleotides of the invention and as matrices, plasmids containing these sequences or derived amplification products. The amplified nucleotide fragments can be used as reagents in hybridization reactions in order to demonstrate the presence, in a biological sample, of a target nucleic acid of sequence complementary to that of said amplified nucleotide fragments. The invention also relates to the nucleic acids capable of being obtained by amplification using primers according to the invention.
Other techniques for amplification of the target nucleic acid can be advantageously used as an alternative to the PCR(PCR-like) using primer pairs of nucleotide sequences according to the invention. By PCR-like is meant any methods implementing direct or indirect reproductions of the nucleic acid sequences, or in which the marking systems have been amplified, these techniques being of course known. Generally, it is a matter of amplification of the DNA by a polymerase; when the original sample is an RNA, reverse transcription should be carried out beforehand. There are currently very numerous processes allowing this amplification, for example the SDA (Strand Displacement Amplification) technique (Walker et al., 1992), the TAS (Transcription-based Amplification System) technique described by Kwoh et al. (1989), the 3SR (Self-Sustained Sequence Replication) technique described by Guatelli et al. (1990), the NASBA (Nucleic Acid Sequence Based Amplification) technique described by Kievitis et al. (1991), the TMA (Transcription Mediated Amplification) technique, the LCR (Ligase Chain Reaction) technique described by Landegren et al. (1988), the RCR (Repair Chain Reaction) technique described by Segev (1992), the CPR (Cycling Probe Reaction) technique described by Duck et al. (1990), the Q--replicase amplification technique described by Miele et al. (1983). Certain of these techniques have since been perfected.
In the case where the target polynucleotide to be detected is an mRNA, it is advantageous to use, prior to the implementation of an amplification reaction using primers according to the invention, or the implementation of a detection process using probes of the invention, a reverse transcriptase type enzyme in order to obtain a cDNA from the mRNA contained in the biological sample. The cDNA obtained will then serve as a target for the primers or probes used in the amplification or detection process according to the invention.
The probes hybridization technique can be carried out in various ways (Matthews et al. 1988). The most general method consists of immobilizing the nucleic acid extracted from the cells of different tissues or cells in culture on a support (such as nitrocellulose, nylon, polystyrene) in order to produce for example DNA chips, then incubating, under well defined conditions, the target nucleic acid immobilized with the probe. After the hybridization, the probe in excess is eliminated and the hybrid molecules formed are detected by the appropriate method (measurement of the radioactivity, fluorescence or enzyme activity linked to the probe).
According to another method of using nucleic probes according to the invention, the latter can be used as capture probes. In this case, a probe, called a capture probe, is immobilized on a support and serves to capture by specific hybridization the target nucleic acid obtained from the biological sample to be tested and the target nucleic acid is then detected using a second probe, called a detection probe, marked by an easily detectable element.
Among the fragments of useful nucleic acids, there should moreover be mentioned in particular the anti-sense oligonucleotides, i.e. the structure of which ensures, by hybridization with the target sequence, an inhibition of the expression of the corresponding product. Sense oligonucleotides should also be mentioned, which, by interaction with proteins involved in the regulation of the expression of the corresponding product, will induce either an inhibition, or an activation of this expression. The oligonucleotides according to the invention have a minimum size of 9 bases, preferably at least 10, 12, 15, 17, 20, 25, 30, 40, 50 bases.
The probes, primers and oligonucleotides according to the invention can be marked directly or indirectly by a radioactive or non-radioactive compound, by methods well known to a person skilled in the art, in order to obtain a detectable and/or quantifiable signal. The non-marked polynucleotide sequences according to the invention can be used directly as probes or primers.
The sequences are generally marked in order to obtain sequences which can be used for numerous applications. The marking of the primers or probes according to the invention is carried out by radioactive elements or by non-radioactive molecules. Among the radioactive isotopes used, there can be mentioned 32P, 33P, 35S, 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: (i), 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: (i) a pair of nucleic primers according to the invention, (ii) the reagents necessary to carry out a DNA amplification reaction, and optionally (iii) a component making it possible to verify the sequence of the amplified fragment, more particularly a probe according to the invention.
The invention also comprises a method for detection and/or assay of a nucleic acid according to the invention, in a biological sample, characterized in that it comprises the following stages: (i) bringing a polynucleotide according to the invention into contact with a biological sample; (ii) detection and/or assay of the hybrid formed between said polynucleotide and the nucleic acid of the biological sample. A subject of the invention is also to provide a kit for the detection and/or assay of a nucleic acid according to the invention, in a biological sample, characterized in that it comprises the following elements: (i) 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 vectors according to the invention comprise the elements necessary for the expression and in particular, preferably a promoter, of the translation initiation and termination signals, as well as appropriate regions for regulation of the transcription. They must be able to be maintained in stable fashion in the cell and can optionally have particular signals specifying the secretion of the translated protein.
The different control signals are chosen as a function of the host cell used. To this end, the nucleic acid sequences according to the invention can be inserted into autonomous replication vectors inside the chosen host, or integrative vectors of the chosen host. Among the autonomous replication systems, as a function of the host cell, plasmid, cosmid, phagemid or mini-chromosome 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 chromosomes (BACs), yeast artificial chromosomes (YACs) for expression in yeast, mouse artificial chromosomes (MACs) for expression in murine cells and in preferred manner human artificial chromosomes (HACs) for expression in human cells.
Such vectors are prepared according to the methods currently used by a person skilled in the art and the resultant clones can be introduced into an appropriate host by standard methods, such as for example lipofection, electroporation, heat shock, transformation after chemical permeabilization of the membrane, cell fusion.
The invention comprises moreover the host cells, in particular the eukaryotic and prokaryotic cells transformed by the vectors according to the invention. Among the cells which can be used within the meaning of the present invention, bacteria and yeasts can be mentioned. According to a preferred embodiment of the invention, the bacterium is chosen from the group composed of Lactobacillus fermentum, Lactobacillus acidophilus, Lactobacillus amylovorus, Lactobacillus crispatus, Lactobacillus helveticum, Lactobacillus lactis, Escherichia coli, Bacillus subtilus, Campylobacter pylori, Helicobacter pylori, Agrobacterium tumefaciens, Staphylococcus aureus, Thermophilus aquaticus, Azorhizobium caulinodans, Rhizobium leguminosarum, Neisseria gonorrhoeae, Neisseria meningitis. According to a preferred embodiment of the invention, the bacterium is Lactobacillus. According to a preferred embodiment it is:
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 N-deoxyribosyltransferases according to the invention is either naturally present in said cell or is introduced into said cell by the recombinant DNA techniques known to a person skilled in the art. According to a preferred embodiment, the nucleic acid sequence introduced into said cell by the recombinant DNA techniques and which codes for an N-deoxyribosyltransferase according to the invention is heterologous. By heterologous nucleic acid sequence is meant a nucleic acid sequence which is not naturally present in the cell according to the invention.
The present invention also relates to metazoic, plant or animal organisms, preferably mammals, except humans, comprising one of said cells transformed according to the invention. Among the animals according to the invention, rodents are preferred, in particular mice, rats or rabbits, expressing at least one polypeptide according to the invention.
The cells, preferably bacterial, or fungal, in particular of yeast, as well as the metazoic organisms according to the invention can be used in a method for producing N-deoxyribosyltransferase according to the invention. The method for producing a polypeptide of the invention in recombinant form, itself included in the present invention, is characterized in that the transformed cells are cultured, in particular the cells of the present invention, under conditions allowing the expression and optionally the secretion of a recombinant polypeptide coded by a nucleic acid sequence according to the invention, and said recombinant polypeptide is recovered. The recombinant polypeptides capable of being obtained by this production method also form part of the invention. They can be presented in glycosylated or non-glycosylated form, and may or may not have the tertiary structure of the natural protein. The recombinant polypeptide sequences can also be modified in order to improve their solubility, in particular in aqueous solvents. Such modifications are known to a person skilled in the art such as for example the deletion of hydrophobic domains or the substitution of hydrophobic amino acids by hydrophilic amino acids.
These polypeptides can be produced from the nucleic acid sequences defined above, according to the techniques for producing recombinant polypeptides known to a person skilled in the art. In this case, the nucleic acid sequence used is placed under the control of signals allowing its expression in a host cell.
An effective system for producing a recombinant polypeptide requires a vector and a host cell according to the invention. These cells can be obtained by the introduction into host cells of a nucleotide sequence inserted into a vector as defined above, then the culture of said cells under conditions allowing the replication and/or expression of the transfected nucleotide sequence.
The processes used for purifying a recombinant polypeptide are known to a person skilled in the art. The recombinant polypeptide can be purified starting with lysates and cell extracts, culture medium supernatant, by methods used individually or in combination, such as fractionation, chromatography methods, immunoaffinity techniques using specific monoclonal or polyclonal antibodies, etc. A preferred variant consists of producing a recombinant polypeptide fused to a carrier protein (chimera protein). The advantage of this system is that it allows a stabilization and reduction of the proteolysis of the recombinant product, an increase in solubility during in vitro renaturation and/or simplification of the purification when the fusion partner has an affinity for a specific ligand.
The polypeptides according to the present invention can also be obtained by chemical synthesis using one of the numerous known peptide syntheses, for example the techniques using solid phases or techniques using partial solid phases, by condensation of fragments or by a synthesis in standard solution. The polypeptides obtained by chemical synthesis and being able to comprise corresponding non-natural amino acids are also included in the invention.
The polypeptides according to the invention make it possible to prepare monoclonal or polyclonal antibodies. It is therefore also one of the subjects of the present invention to provide a monoclonal or polyclonal antibody and its fragments, characterized in that they selectively and/or specifically bind a polypeptide according to the invention. The chimeric antibodies, humanized antibodies and single-chain antibodies also form part of the invention. The antibody fragments according to the invention are preferably Fab, F(ab)2, Fc or Fv fragments. The polyclonal antibodies can be prepared, for example by immunization of an animal, in particular a mouse, with a polypeptide according to the invention combined with an immune response adjuvant, then purification of the specific antibodies contained in the serum of the animals immunized on an affinity column on which the polypeptide having served as antigen has been fixed beforehand. The polyclonal antibodies according to the invention can also be prepared by purification on an affinity column on which a polypeptide according to the invention has been immobilized beforehand. The monoclonal antibodies can advantageously be prepared from hybridomas according to the technique described by Kohler and Milstein in 1975.
According to a particular embodiment of the invention, the antibody is capable of inhibiting the interaction between the N-deoxyribosyltransferase of the invention and its substrate in order to alter the physiological function of said N-deoxyribosyltransferase polypeptide.
The invention also relates to methods for the detection and/or purification of a polypeptide according to the invention, characterized in that they use an antibody according to the invention. The invention moreover comprises purified polypeptides, characterized in that they are obtained by a method according to the invention.
Moreover, apart from their use for the purification of the polypeptides, the antibodies of the invention, in particular the monoclonal antibodies, can also be used for the detection of these polypeptides in a biological sample.
For these different uses, the antibodies of the invention can also be marked in the same manner as described previously for the nucleic probes of the invention and in preferred manner with an enzymatic, fluorescent or radioactive marking.
The antibodies of the invention also constitute a means of analysis of the polypeptide expression according to the invention, for example by immunofluorescence, marking with gold, enzyme immunoconjugates. More generally, the antibodies of the invention can be advantageously used in any situation where the expression of a polypeptide according to the invention, normal or mutated, must be observed, and more particularly in immunocytochemistry or immunohistochemistry or in western blotting experiments. Thus, a process for detecting a polypeptide according to the invention in a biological sample, comprising the stages of bringing the biological sample into contact with an antibody according to the invention and demonstrating the antigen-antibody complex formed is also a subject of the invention.
It is also one of the subjects of the present invention to provide a process for in vitro or in vivo enzymatic synthesis of deoxyribonucleotides characterized in that it comprises at least one reaction stage catalyzed by at least one N-deoxyribosyltransferase according to the invention. The process according to the invention is characterized in that said N-deoxyribosyltransferase catalyzes the exchange of a first nucleobase present in a deoxyribonucleoside by a second nucleobase.
According to a preferred embodiment of the invention, said second nucleobase is selected from the group composed of the purines bound by N9, pyrimidines bound by N1, azines bound by N1, imidazoles bound by N1, said second nucleobases being able to carry substitutions of the hydrogens at the non-bound positions. Preferably, said second nucleobase is selected from the group composed of 6-methyl purine, 2-amino-6-methylmercaptopurine, 6-dimethylaminopurine, 5-azacytidine, 2,6-dichloropurine, 6-chloroguanine, 6-chloropurine, 6-aza-thymine, 5-fluoro-uracile, ethyl-4-amino-5-imidazole carboxylate, imidazole-4-carboxamide and 1,2,4-triazole-3-carboxamide.
Said first nucleobase is itself preferably selected from the group composed of adenine, guanine, thymine, uracile and hypoxanthine. These lists are not exhaustive, and it is evident that natural or non-natural analogues of nucleobases can be used in the present invention as substrate of an N-deoxyribosyltransferase of the invention.
Optionally, the in vivo process according to the invention is characterized in that it moreover comprises the stage of introducing into the host cell the first nucleobase present in a deoxyribonucleoside.
Optionally, the in vivo process according to the invention is characterized in that it moreover comprises the stage of introducing into the host cell the second nucleobase present in a deoxyribonucleoside.
Optionally, the in vivo process according to the invention is characterized in that it moreover comprises the stage of introducing into the host cell the first nucleobase present in a deoxyribonucleoside and the second nucleobase simultaneously and/or one after the other.
The deoxyribonucleosides capable of being produced in large quantities and inexpensively by the biosynthesis method according to the invention therefore constitute compounds of interest intended for the preventive or curative treatment of human or animal, tumorous, viral pathologies such as AIDS (acquired human immunodeficiency syndrome), bacterial, parasitic or fungal pathologies. Alternatively, these deoxyribonucleosides capable of being produced in large quantities and inexpensively by the biosynthesis method according to the invention constitute herbicides and insecticides.
The present invention also provides a process for nutritional screening intended to isolate deoxyribosyltransferases, preferably the polypeptides according to the invention but also their homologues or their mutants. This first screening according to the invention comprises the stages:
Other characteristics and advantages of the invention are clear from the rest of the description, with examples represented hereafter.
1.1 Strains and Culturing Conditions
The strains of lactic bacteria used originate from the CNRZ (Centre National de Recherche Zootechnique) collection, Unit de Recherches Laitires et Gntique Applique, INRA, Jouy en Josas. They are cultured in MRS medium (from Man et al., J. Appl. Bacteriol., 23: 130-135, 1960) and incubated at 30 C., 37 C. or 42 C. according to the species. The Escherichia coli TG1 strain, provided by Stratagne, is cultured in LB (Luria broth base 10 g/L, Agar-agar 16 g/L) under agitation and at 37 C.
1.2. Preparation of Total Cellular DNA of Lactic Bacteria:
The cultures at the end of the exponential phase are centrifuged for 5 minutes at 13,000 g. The pellet corresponding to a culture of 2 ml is taken up in 200 l of TES (50 mM Tris, pH8, 10 mM EDTA, pH8, 250 mM saccharose) containing 20 g/ml of lysozyme and 50 U/ml of mutanolysine (Sigma). After an incubation of one hour at 37 C., the clarification of the preparation is obtained by adding 60 l of 20% SDS.
The extraction of the nucleic acids is carried out by adding to the lysate 500 l of saturated phenol in water, pH8, to which 0.1% hydroxyquinoline and 100 l of a mixture of isoamylic chloroform-alcohol (24/l, V/V) has been added. The solution is homogenized then centrifuged for 10 minutes at 13,000 g and at ambient temperature. The upper, limpid phase containing the nucleic acids is retained. The extraction is repeated three times on the latter in order to eliminate the undesired cellular constituents. The phenol traces are eliminated by adding 500 l of isoamylic chloroform-alcohol to the aqueous phase. After homogenization and centrifuging for 3 minutes at 15,000 g and at 4 C., the nucleic acids contained in the upper aqueous phase are precipitated by the addition of a volume of cold isopropanol. After an incubation of one hour at 20 C., a centrifugation is carried out for 20 minutes at 15,000 g and at 4 C. The isopropanol is eliminated and replaced by 500 l of 70% ethanol. A final centrifugation of 10 minutes at 15,000 g and at 4 C. allows a pellet of nucleic acids to be recovered. This is left to dry in an evaporator and resuspended in 200 l of sterile water containing 10 l of RNase at 10 g/l. After 15 minutes of incubation at 37 C. to agitate the enzyme degrading the RNAs, 10 l 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 l containing 20 to 100 ng of DNA, 0.5 M primers, 200 M dNTPs (dATP, dCTP, dGTP, dTTP) in a 10 mM Tris-HCl buffer pH 9, 50 mM KCl, 1.5 mM MgCl2, 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 ntd1 (SEQ ID no. 15) and ntd2 primers (SEQ ID no. 16) starting from the ntd sequence of Lactobacillus leichmanii described by Huck (personal communication):
1.4. Southern-Type Hybridization:
Enzymatic restriction of the DNAs. The total DNAs are digested by one or more restriction enzymes. The enzymes used are: BamHI, BglII, ClaI, EcoRI, HindIII, HpaI, NcoI, NotI, PstI, XbaI, XhoI (Bio-Lab). Digestion is carried out in a final volume of 40 l containing 70 U of enzyme, 4 l of 10NEB buffer (Bio-Lab) and 4 to 8 g of DNA. The incubation is carried out for 1 hour 30 minutes at 37 C.
Transfer of the DNA to a membrane. The total DNA fragments resulting from the enzymatic digestion are separated using a 0.7% agarose gel. After migration, the agarose gel is placed under agitation in a depurination solution (0.25N HCl) 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 NaCl, 0.5M NaOH. The gel is rinsed with water then incubated again for 30 minutes in a neutralization solution, 1.5M NaCl, 0.5M Tris HCl; pH 7.5. The DNAs are transferred by capillarity onto a positively charged nylon membrane (Hybond N+, Amersham). They are eluted by a rising flux of 20SSC (0.3M trisodium citrate; 3M NaCl; pH7). After the transfer, the DNAs are covalently bound onto the membrane using a UV Stratalinker 2400 apparatus (Stratagene).
Preparation of the probe. The Probe used is an internal fragment of the ntd gene of Lactobacillus helveticus amplified by PCR. The probe is purified using the Wizard kit (Promega) in order to eliminate the PCR primers. The necessary concentration of the probe is 10 ng/l. The DNA is marked using the ECL marking kit (Amersham). To do this, the DNA is denatured by heating for 5 minutes at 100 C. and immediately recooled in ice. A volume of marking reagent (peroxidase) then a volume of glutaraldehyde solution are added. This solution is incubated for 10 minutes at 37 C. for covalently binding the peroxidase to the DNA.
Hybridization and development. After a prehybridization of one hour at 42 C. in hybridization buffer, the membrane is hybridized for 16 hours at 42 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 urea0.4% SDS0.5SSC, then rinsed for 5 minutes in two successive baths of buffer: 0.3M sodium citrate3M 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 PCP are inserted into the plasmid vector pBluescript II SK+ of E. coli. TG1 (Stratagene). This plasmid is first restricted in its single site by the EcoRV enzyme (Gibco-BRL) which creates free ends. The digestion mixing is carried out in a volume of 30 l, containing 4 l of DNA. The DNA fragments amplified to be cloned should have their 5 and 3 ends free in order to allow cloning. The preparation of the free ends of 50 l of PCR products purified using the Wizard kit (Promega) is carried out in a reaction volume of 100 l containing 3.6 units of polymerase DNA of the T4 phage (Bio-Lab) and 6 units of polymerase I DNA (or Klenow fragment) (Bio-Lab), not having a 53 exonucleic activity. The polymerization is carried out for 20 minutes at 11 C. then the enzymes are deactivated after 10 minutes at 75 C. The DNA is then precipitated in the presence of two volumes of 100% ethanol, glycogen and 10% 3M sodium acetate, pH 4.8. The mixture is placed for one hour at 20 C. then centrifuged for 20 minutes at 15,000 g. The pellet is rinsed with 250 l of 70% ethanol, centrifuged again for 10 minutes at 15,000 g, dried in an evaporator and resuspended in 20 l 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 l containing 60 ng of insert, 26 ng of restricted pBS-SK+ plasmid, 2 units of ligase (T4 DNA ligase, Boehringer-Mannheim), overnight at 16 C. The ligation products are dialyzed on a 0.025 m millipore filter so as to eliminate the salts and to thus avoid electric arcs during electroporation.
Preparation of electrocompetent cells of the TG1 strain of E. coli. Starting from 5 ml of an overnight culture at 37 C. under agitation, 500 ml of LB medium are inoculated. The culture is placed under agitation at 37 C. until an O.D.600nm=1 is reached. It is then recooled for 2 hours in ice then cold-centrifuged for 10 minutes at 5,000 rpm. The supernatant is eliminated, the pellet is taken up in 400 ml of cold water. This preparation is cold-centrifuged for 10 minutes and at 5,000 rpm. The pellet obtained is taken up again in 250 ml of cold water. Following a centrifugation of 10 minutes, the pellet is taken up in 25 ml of cold water then the cells are suspended in a final volume of 1 ml of 10% glycerol, and aliquotted before being rapidly frozen in liquid nitrogen.
Transformation by electroporation and selection of clones. The electrocompetent cells preserved at 80 C. are thawed in ice then brought into contact with 5 l 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 (20 g/L bactopeptone, 5 g/L yeast extract, 2 ml/L 5M NaCl, 2.5 ml/L 1M KCl, 10 ml/L 1M MgCl2, 10 ml/L 1M MgSO4), containing 0.4% glucose in cell suspension which is incubated at 37 C. for one hour. The cells are then plated on an LB selective mediumXgal (5-bromo-4-chloro-3-indolyl--D-galactoside, 1 ug/ml)IPTG (isopropythio--D-galactoside, lpg/ml)ampicillin (50 g/ml) and incubated at 37 C. overnight.
Rapid extraction of plasmidic DNA of recombinant E. coli clones by alkaline lysis. The E. coli cells transformed and cultivated in LB medium containing ampicillin (100 g/ml) are harvested by centrifugation at 15,000 for 10 minutes at 4 C. They are resuspended in 100 l of a 50 mM saccharose solution, 25 mM Tris-HCl, pH 8, mM EDTA, pH 8. The alkaline lysis and the denaturation of the DNA is carried out by addition of 200 l 0.2N NaOH, 1% SDS. The reaction medium is left for 1 minute at ambient temperature after having added 200 l of chloroform. Then 150 l of a solution of 5M potassium acetate, glacial acetic acid are added. The reaction medium is centrifuged for 15 minutes at 13,000 g at 4 C. The aqueous phase containing the DNA is precipitated in the presence of 2 volumes of 100% ethanol then centrifuged for 20 minutes at 13,000 g at 4 C. The pellet is washed in 70% ethanol, centrifuged for 10 minutes at 13,000 g then resuspended in 30 l 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 Southern-type 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 l of ligation buffer are added to 4 to 8 g of DNA in a final volume of 1 ml. The ligation mixture is incubated at 15 C. overnight. The ligated DNA is then precipitated with 100 l of 3M sodium acetate, pH 4.8, 700 l of isopropanol and 2 l of glycogen, then centrifuged for 30 minutes at 13,000 g at 4 C. The pellet is rinsed in 300 l of 70% ethanol and centrifuged for 10 minutes at 13,000 g at 4 C. The pellet is taken up in 25 l of ultrapure water.
Amplification of circularized DNAs using different primers. The polymerase chain reactions are carried out in a reaction volume of 100 l containing 20 to 100 ng of DNA, 0.5 M primers, 200 M dNTPs (dATP, dCTP, dGTP, dTTP) in a 10 mM Tris-HCl buffer pH 9, 50 mM KCl, MgCl2 with 1.5 mM BSA at 0.002% and 2.5 units of Taq polymerase.
The amplification is carried out under the following conditions:
Sequencing. The PCR fragments are purified by the Wizard kit (Promega) in order to eliminate the non-incorporated oligonucleotides, the salts and the Taq polymerase. The sequencing is carried out using a 373A automatic sequencer (Applied Biosystems-Perkin Elmer) using an ABI PRISM Dye Terminator kit (Perkin Elmer) based on the incorporation of fluorescent phosphate deoxynucleotides during the elongation phase of the primers. The sequence reactions are carried out in a reaction volume of 20 l containing 30 ng of DNA, 4 l of DyeT Mix (Perkin Elmer Biosystems) and 0.1 mM of primer.
20 l of 3M sodium acetate, pH 4.6, 50 l of 95% ethanol and 1 l 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 l of 70% ethanol then centrifuged for 10 minutes at 13,000 g. The pellet is then taken up in 6 l 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 l deposited on 4% acrylamide gel.
A functional screening allowing the production of guanine to be selected was established on E. coli by deleting the two genes of the guaBA operon which controls the conversion of IMP into XMP then into GMP and also those of the deoCABD operon which controls the degradation of deoxynucleosides to give the PAK 6 strain. The E. coli genome specifies an activity allowing the base G to be converted into GMP (guanine phosphoribosyltransferase encoded by the gpt gene), and also an activity allowing the base G to be released from the dR-G deoxynucleoside (purine nucleoside phosphorylase encoded by the deoD gene in the deo operon). The PAK 6 strain thus has a requirement for guanine (G) which cannot be satisfied by the addition of deoxyguanosine (dR-G). The use of deoxyguanosine (dR-G) can however be selected if an N-deoxyribosyltransferase activity is expressed in the PAK6 strain in order to carry out the exchange: dR-G+A-dR-A+G.
This exchange between two purine bases can be catalyzed by the two enzyme classes. In fact, the introduction of the ntd gene of L. leichmannii into the PAK 6 strain allows the requirement for guanine to be satisfied using deoxyguanosine (dR-G) and adenine (A).
DNA fragments of a size comprised between 1 and 2 kb obtained by partial digestion (AluI) 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 (A) 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 N-deoxyribosyltransferase activity and deviating from the restriction profile of the ntd gene of L. helveticus. The sequence of this insert develops a gene specifying a polypeptide of 167 amino acids with a molecular weight of 18790.70 Daltons having a similarity of 28.6% with the NTD polypeptide of L. leichmanii. The sequence of this gene called ptd deviates from that of the ntd genes making them impossible to hybridize (7.5% identity).
Incidentally the ntd gene of L. helveticus controlling a Class II N-deoxyribosyltransferase activity could be isolated once again among the transforming clones selected.
The same nutritional cloning and selection operations were carried out starting from genomic DNA of the L. fermentum CIP 102980T strain. The transforming clones selected proved to propagate a plasmid the inserts of which, having similar restriction profiles, controlled a Class II N-deoxyribosyltransferase activity. The sequence of one of these inserts developed a gene specifying a polypeptide of 168 amino acids with a molecular weight of 18878.20 Daltons having a similarity of 32.9% with the NTD polypeptide of L. leichmanii and 36.7% with the PTD polypeptide of L. helveticus. The sequence of this gene deviates from that of the ntd and ptd genes which makes them impossible to hybridize. The NTD polypeptide of L. fermentum has a more marked evolutive relationship for the enzyme of Class I (PTD of L. helveticus) and a functional affinity to Class II, suggesting an early evolutive divergence in the evolution of these enzymes. Its enzymatic activity could prove to be different to those of other species and be suitable for the preparation of a very large spectrum of nucleosides.
Using degenerated oligonucleotides starting from regions of the amino acid sequence of the NTD polypeptide of L. leichmanii (Hck, 1997) an internal fragment on the ntd gene of L. helveticus was amplified. Starting from this fragment, oligonucleotides were synthesized so as to obtain all the gene by inverse PCR.
Starting from the two ntd sequences of L. leichmannii and L. helveticus, we redefined the consensus primers by isolating the ntd genes from other species of lactobacilli such as L. acidophilus, L. crispatus, L. amylovorus with the same result as that described above.
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+TdT+G. Only the colonies expressing an N-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 N-deoxyribosyltransferase II (ntd) activity to be correlated with the pLH2 plasmid containing the polynucleotide of SEQ ID No. 1 and coding for the ntd enzyme of lactobacillus helveticus and the N-deoxyribosyltransferase I (ptd) activity with the pLH4 plasmid containing the polynucleotide of SEQ ID No. 3 and coding for the ptd enzyme of lactobacillus helveticus.