EP1499713A2 - Genbank von cyanophage s-2l und deren funktionsanalyse - Google Patents

Genbank von cyanophage s-2l und deren funktionsanalyse

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Publication number
EP1499713A2
EP1499713A2 EP03747467A EP03747467A EP1499713A2 EP 1499713 A2 EP1499713 A2 EP 1499713A2 EP 03747467 A EP03747467 A EP 03747467A EP 03747467 A EP03747467 A EP 03747467A EP 1499713 A2 EP1499713 A2 EP 1499713A2
Authority
EP
European Patent Office
Prior art keywords
polypeptide
nucleotide sequence
bases
sequence
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03747467A
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English (en)
French (fr)
Inventor
Philippe Marliere
Pierre-Alexandre Kaminski
Frédérique GALISSON
Madeleine Bouzon
Sylvie Pochet
Jean Weissenbach
William Saurin
Catherine Robert
Virginie Vico
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Centre National de la Recherche Scientifique CNRS
Commissariat a lEnergie Atomique et aux Energies Alternatives CEA
Institut Pasteur
Original Assignee
Genoscope - Centre National De Sequencage
Centre National de la Recherche Scientifique CNRS
Institut Pasteur
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Application filed by Genoscope - Centre National De Sequencage, Centre National de la Recherche Scientifique CNRS, Institut Pasteur filed Critical Genoscope - Centre National De Sequencage
Publication of EP1499713A2 publication Critical patent/EP1499713A2/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C07D473/02—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6
    • C07D473/16—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6 two nitrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the subject of the present invention is the genomic sequence and nucleotide sequences coding for polypeptides of the cyanophagus S-2L.
  • the polypeptides described in the present invention are, without limitation, polypeptides involved in the synthesis, transcription and replication of purine bases.
  • the determination of the genome of cyanophagus S-2L is a useful tool for the supply of genes, which expressed in recombinant bacteria, allow the synthesis of DNA monomers incorporating the D base (2.6 diaminopurine) instead of base A (adenine) and thus produce chemically reshaped nucleic acids in bacteria.
  • the invention also relates to the use of the genomic sequence and / or the nucleotide and / or polypeptide sequences described in the present invention for the analysis of gene expression.
  • the two main nucleic acids DNA and RNA are polymers of nucleotides which are composed of a purine or pyrimidine base linked to a 5-carbon sugar (deoxyribose in the case of DNA, ribose in TARN) by an N-glycosidic bond. and a phosphate esterified to the hydroxyl group of the carbon located at the 5 ′ position of the sugar.
  • RNA and DNA contain four types of nucleotides which are distinguished by their bases: adenine (A), guanine (G), cytosine (C) and uracil (U) for RNA; 5-methyluracil, i.e. thymine (T), replacing uracil in DNA.
  • A adenine
  • G guanine
  • C cytosine
  • U uracil
  • T replacing uracil in DNA
  • Modified bases are observed in the DNA of all organisms, and may be involved in phenomena regulating gene expression (5). Except in bacteriophages, the DNA modifications known up to now are produced by post-replicative enzymatic reactions, of which a DNA duplex is the substrate. On the contrary, during infection with certain bacteriophages, the DNA modifications known up to now are produced by pre-replicative enzymatic reactions, of which a nucleotide is the substrate, to lead to a non-canonical deoxynucleoside triphosphate.
  • the entities include: dUTP, 5-hydroxymethyl-dUTP, 5-dihydroxypentyl-dUTP, 5-hydroxymethyl-dCTP. Another entity is strongly suspected: 5-methyl-dCTP (11).
  • the emergence of modified bases in bacteriophages is generally interpreted as a countermeasure to the restriction systems of bacteria (11).
  • Bromouracil or 8-azaguanine are synthetic analogs of the natural bases thymine and guanine. These analogs are converted to triphosphate nucleotides by the purine or pyrimidine backup pathways and are then incorporated into DNA.
  • 6-methyladenine and 5-methylcytosine are the most frequently encountered modified bases.
  • Methylated nucleotides are not incorporated as such into DNA but are the product of the action of specific DNA methyltransferases. These enzymes transfer the methyl group from S-adenosylmethionine to adenine or cytosine after DNA replication.
  • DNA methyltransferases transfer the methyl group from S-adenosylmethionine to adenine or cytosine after DNA replication.
  • DNA methylation influences the regulation of gene expression and cell differentiation.
  • T phages such as bacteriophage T4
  • cytosine is systematically replaced by 5-hydroxymethylcytosine. This substitution requires on the one hand a pathway for the biosynthesis of hydroxymethyldoxycytidine triphosphate (HM dCTP) as well as enzymes allowing the exclusion of the normal base.
  • HM dCTP hydroxymethyldoxycytidine triphosphate
  • the HMC DNA biosynthesis pathway involves a hydroxymethylase which converts dCMP to hydroxymethyl dCMP, a nucleoside monophosphate kinase which phosphorylates HM dCMP to give diphosphate, precursor of HM dCTP which is then incorporated into DNA polymerase then glycosylated by a gly cozy ltransferase.
  • Excluding cytosine involves endonucleases on the one hand specific for DNA containing this base and a dCDPase-dCTPase which converts the corresponding nucleotides into dCMP which is then the substrate for dCMP hydroxymethylase and dCMP deaminase.
  • DCMP deaminase generates the dUMP precursor of dTMP.
  • thymine is replaced by 5-hydroxymethyluracil (phages SPO1 and ⁇ e) or uracil (phages PBS2) in several phages of Bacillus substilis (Warren, 1980; Kornberg and Baker, 1991 ).
  • phages such as SP15 or ⁇ W14 have DNA whose thymine has been replaced by 5-dihydroxypentyluracil and -putrescinylthymine. However, this replacement is only partial and seems to be due to post-replicative modifications.
  • Cyanophagus S-2L was isolated from water samples collected in the Leningrad region. This phage is capable of lyzing a relatively small number of Synechococcus: sp. 698, 58 and PCC6907. From a morphological point of view it consists of an icosahedral head and a flexible non-contractile tail. S-2L is said to be part of a family whose other member could be phage SM-2 which is morphologically similar to it (Fox et al. 1976). The phage S-2L DNA is linear double strand with a size of 42 kb composed of
  • the invention aims in particular to sequence the genome of phage S-2L, so as to obtain a deposit of genes which, once propagated in isolation and expressed under control in recombinant bacteria, are intended in particular to form biotechnologically new monomers DNA and to produce, see replicate, chemically reshaped nucleic acids in bacteria.
  • the invention also aims to use nucleotide sequences obtained for the identification of metabolic pathways leading to the production of D bases.
  • the invention also relates to the enzymatic production of deoxynucleoside analogues which are very useful in particular in the chemotherapy of AIDS.
  • the invention also aims to express in a cyanophagous S2L host nucleic acids coding for proteins involved in the metabolism of D bases.
  • the invention also aims to obtain S-2L genes which propagated individually in E. coli and expressed under strict transcriptional control will make it possible to test the hypotheses concerning their function in the metabolism of the nucleotides, the replication and the transcription.
  • the invention relates, according to a first aspect, to a nucleotide sequence of the cyanophagus S-2L corresponding to SEQ ID No. 1.
  • the present invention also relates to a nucleotide sequence of cyanophagus S-2L chosen from: a) a nucleotide sequence comprising at least 80%, 85%, 90%, 95% or 98% identity with SEQ ID No. 1; b) a nucleotide sequence hybridizing under conditions of high stringency with SEQ ID No. 1; c) a nucleotide sequence complementary to SEQ ID
  • the present invention also relates to the nucleotide sequences characterized in that they come from SEQ ID No. 1 and in that they code for polypeptides chosen from the sequences SEQ ID No. 2 to SEQ ID No. 527 or a biologically active fragment of these polypeptides.
  • the invention also relates to the nucleotide sequences characterized in that they comprise a nucleotide sequence chosen from: a) a nucleotide sequence derived from SEQ ID No. 1 and coding for a polypeptide chosen from the sequences from SEQ ID No. 2 to SEQ ID N °
  • nucleotide sequence comprising at least 80%, 85%, 90%, 95% or 98% of identity with a nucleotide sequence according to a); c) a nucleotide sequence hybridizing under conditions of high stringency with a nucleotide sequence according to a) or b); d) a complementary nucleotide or RNA sequence corresponding to a sequence as defined in a), b) or c); e) a nucleotide sequence of a fragment representative of a sequence as defined in a), b), c) or d); and f) a modified nucleotide sequence of a sequence as defined in a), b), c), d) or e),
  • the invention relates to a nucleotide sequence characterized in that it codes for a polypeptide chosen from: a) the polypeptides of cyanophagus S-2L of sequences SEQ ID No. 2 to SEQ ID NO: a) the polypeptides of cyanophagus S-2L of sequences SEQ ID No. 2 to SEQ ID NO: a) the polypeptides of cyanophagus S-2L of sequences SEQ ID No. 2 to SEQ ID
  • polypeptides having at least 80%, preferably 85%, 90%, 95% and 98% identity with a polypeptide of a), b), c); e) the biologically active fragments of the polypeptides of a), b), c), d) f) the modified polypeptides of a), b), c), d), e).
  • the invention also relates to a nucleotide sequence characterized in that it comprises a nucleotide sequence chosen from: a) a nucleotide sequence as defined above; b) a nucleotide sequence comprising at least 80% identity with a nucleotide sequence of a); c) a nucleotide sequence hybridizing under conditions of high stringency with a nucleotide sequence of a) or b); d) a complementary nucleotide or RNA sequence corresponding to a sequence as defined in a), b) or c); e) a nucleotide sequence of a fragment representative of a sequence as defined in a), b), c) or d); and f) a nucleotide sequence modified from a sequence as defined in a), b), c), d) or e).
  • nucleic acid nucleic or nucleic acid sequence, polynucleotide, oligonucleotide, polynucleotide sequence, nucleotide sequence, terms which will be used interchangeably in the present description, is intended to denote a precise sequence of nucleotides, modified or not, making it possible to define a fragment or region of a nucleic acid, which may or may not contain unnatural nucleotides, and which may correspond to both double-stranded DNA, single-stranded DNA and transcripts of said DNAs.
  • the nucleic acid sequences according to the invention also include PNA (Peptid Nucleic Acid), or the like.
  • nucleotide sequences in their natural chromosomal environment that is to say in the natural state.
  • sequences which have been isolated and / or purified that is to say that they have been taken directly or indirectly, for example by copying, their environment having been at least partially modified.
  • This also means the nucleic acids obtained by chemical synthesis.
  • percentage of identity between two nucleic acid or amino acid sequences within the meaning of the present invention is meant a percentage of identical nucleotides or amino acid residues between the two sequences to be compared, obtained after the best alignment, this percentage being purely statistical and the differences between the two sequences being distributed at random and over their entire length.
  • the term “best alignment” or “optimal alignment” is intended to denote the alignment for which the percentage of identity determined as below is the highest. Sequence comparisons between two nucleic acid or amino acid sequences are traditionally carried out by comparing these sequences after having optimally aligned them, said comparison being carried out by segment or by "comparison window” to identify and compare the regions. sequence similarity locale.
  • the optimal alignment of the sequences for comparison can be achieved, besides manually, by means of the local homology algorithm of Smith and Waterman (1981, Ad. App. Math. 2: 482), by means of the algorithm. of local homology by Neddleman and Wunsch (1970, J. Mol. Biol. 48: 443), using the similarity search method of Pearson and Lipman (1988, Proc. Natl. Acad. Sci. USA 85: 2444 ), using computer software using these algorithms (GAP, BESTFIT, BLAST P, BLAST N, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI).
  • the BLAST program is preferably used with the BLOSUM 62 matrix.
  • the PAM or PAM250 matrices can also be used.
  • the percentage of identity between two nucleic acid or amino acid sequences is determined by comparing these two optimally aligned sequences in which the nucleic acid or amino acid sequence to be compared may include additions or deletions compared to the reference sequence for optimal alignment between these two sequences.
  • the percentage of 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 to obtain the percentage of identity between these two sequences.
  • nucleic acid sequences having a percentage identity of at least 80%, preferably 85% or 90%, more preferably 95% or even 98%, after optimal alignment with a reference sequence is meant the nucleic acid sequences having , with respect to the reference nucleic acid sequence, certain modifications such as in particular a deletion, a truncation, an elongation, a chimeric fusion and / or a substitution, in particular punctual, and whose nucleic sequence has at least 80%, preferably 85%, 90%, 95% or 98%, identity after optimal alignment with the reference nucleic sequence.
  • They are preferably sequences whose complementary sequences are capable of hybridizing specifically with the reference sequences.
  • the specific hybridization conditions or high stringency will be such that they ensure at least 80%, preferably 85%, 90%, 95% or 98% of identity after optimal alignment between one of the two sequences and the complementary sequence of the other.
  • Hybridization under conditions of high stringency means that the conditions of temperature and ionic strength are chosen in such a way that they allow hybridization to be maintained between two complementary DNA fragments.
  • high stringency conditions of the hybridization step for the purpose of defining the polynucleotide fragments described above are advantageously as follows.
  • DNA-DNA or DNA-RNA hybridization is carried out in two stages: (1) prehybridization at 42 ° C for 3 hours in phosphate buffer (20 mM, pH 7.5) containing 5 x SSC (1 x SSC corresponds to a 0.15 M NaCl + 0.015 M sodium citrate solution), 50% formamide, 7% sodium dodecyl sulfate (SDS), 10 x Denhardt's, 5% dextran sulfate and 1% salmon sperm DNA; (2) actual hybridization for 20 hours at a temperature depending on the size of the probe (ie: 42 ° C, for a probe of size> 100 nucleotides) followed by 2 washes of 20 minutes at 20 ° C in 2 x SSC + 2% SDS, 1 wash for 20 minutes at 20 ° C in 0.1 x SSC + 0.1% SDS.
  • the last washing is carried out in 0.1 ⁇ SSC + 0.1% SDS for 30 minutes at 60 ° C. for a probe of size> 100 nucleotides.
  • the conditions of high stringency hybridization described above for a polynucleotide of defined size can be adapted by the skilled person for oligonucleotides of larger or smaller size, according to the teaching of Sambrook et al., ( 1989, Molecular cloning: a laboratory manual. 2 nd Ed. Cold Spring Harbor).
  • fragment representative of sequences according to the invention is intended to denote any nucleotide fragment having at least 15 nucleotides, preferably at least 20, 30, 75, 150, 300 and 450 consecutive nucleotides of the sequence from which it is derived.
  • ORFs sequences ORFs for "Open Reading Frame"
  • the numbering of the nucleotide sequences ORFs which will be used subsequently in the present description corresponds to the numbering of the amino acid sequences of the proteins encoded by said ORFs.
  • nucleotide sequences ORF2, ORF3 ..., ORF526 and ORF527 respectively code for the proteins of amino acid sequences SEQ ID N ° 2, SEQ ID N ° 3 ..., SEQ ID N ° 526 and SEQ ID N ° 527 appearing in the sequence list of the present invention.
  • the detailed nucleotide sequences of the sequences ORF2, ORF3 ..., ORF526 and ORF527 are determined by their respective positions on the genomic sequence SEQ ID No. 1 of cyanophagus S2L. Table 1 provides the coordinates of 54 preferred ORFs with respect to the nucleotide sequence SEQ ID No.
  • sequence listing indicates for each of the 526 ORFs identified, numbered ORF2 to ORF527, the reading frame.
  • sequence SEQ ID No. 1 is a DNA strand in the 5 ′ -3 ′ orientation
  • sequence SEQ ID No. 2 is a protein sequence encoded by ORF No. 2.
  • a “positive” frame of +1 corresponds to the reading frame designated +1 starting at nucleotide nt 3 of SEQ ID No. 1 ( 1st codon of 1ORF2 located on this reading frame and starting at nt 9 of SEQ ID No. l: TCG which corresponds to serine S; 2 nd codon of ORF 2 according to this framework: GAG which corresponds to glutamic acid E).
  • a +2 frame corresponds to the reading frame designated +2 and starting at nucleotide nt 1 of SEQ ID No. 1 ( 1st codon of ORF
  • a +3 frame corresponds to the designated reading frame +3 starting at nucleotide nt 2 of SEQ ID No.1 ( 1st codon of ORF 5 located on this reading frame and starting at nt35 of SEQ ID No.1: CGT which corresponds to arginine R; 2 nd codon of ORF 5 according to this framework: TCA which corresponds to serine
  • ORF 2 starts at nt n ° 9 of SEQ ID n ° l (ie base T) and stops at nt n ° 515 (ie base G).
  • ORF 4 starts at nt 10 of SEQ
  • a negative frame corresponds to the complementary antiparallel strand of the positive strand.
  • the sequence on the complementary strand TAC will read CAT.
  • the complementary strand of nucleotides 782 to 791 (CCT CGA TAG) is (GGA GCT ATC) reading in the negative direction CTA TCG AGG which correspond respectively to amino acids L , S, R.
  • the representative fragments according to the invention can be obtained for example by specific amplification such as PCR or after digestion with appropriate restriction enzymes of nucleotide sequences according to the invention, this method being described in particular in work by Sambrook et al. Said representative fragments can also be obtained by chemical synthesis when their size is not too large, according to methods well known to those skilled in the art.
  • sequences containing sequences of the invention we also mean the sequences which are naturally framed by sequences which have at least 80%, 85%, 90%, 95% or 98% identity with the sequences according to the invention.
  • modified nucleotide sequence any nucleotide sequence obtained by mutagenesis according to techniques well known to those skilled in the art, and comprising modifications with respect to the normal sequences, for example mutations in the regulatory and / or promoter sequences of the expression of the polypeptide, in particular leading to a modification of the level of expression or of the activity of said polypeptide.
  • modified nucleotide sequence is also meant any nucleotide sequence coding for a modified polypeptide as defined below.
  • the present invention provides all the nucleotide and polypeptide sequences of the cyanophagous S-2L genome. Furthermore, it is an object of the present invention to disclose the functions of these genes and proteins.
  • the genes described in the invention were isolated from DNA fragments using primers deduced from the sequence of the cyanophagus S-2L.
  • the invention relates to a nucleotide sequence characterized in that it codes for a cyanophagous polypeptide S-2L or one of its representative fragments involved in the metabolism of nucleotides, purines, pyrimidines or nucleosides.
  • the term "representative agent" for a peptide means a biologically active fragment of this peptide (having an activity of at least 10, 20, 50, 100% of the activity obtained with this peptide).
  • the invention relates to a nucleotide sequence characterized in that it codes for a cyanophagous polypeptide S-2L or one of its representative fragments involved in the metabolism of base D nucleotides, in particular a peptide of sequence SEQ ID No. 175 or one of its representative fragments.
  • the invention relates to a nucleotide sequence characterized in that it codes for a cyanophagous polypeptide S-2L or one of its representative fragments involved in the replication process, in particular a peptide of sequence SEQ ID No. 14,18,142,355,429,454 or one of their representative fragments.
  • the invention relates to a nucleotide sequence characterized in that it codes for an envelope polypeptide, in particular of capsid, of cyanophagus S-2L or one of its representative fragments, in particular a peptide of sequence SEQ ID No. 169,316,351,392,395,406,422,425 or one of their representative fragments.
  • the invention relates to a nucleotide sequence according to the invention characterized in that it codes for a cyanophagous polypeptide S-2L or one of its fragments involved in the diversion of cellular machinery.
  • the invention relates to a nucleotide sequence according to the invention characterized in that it codes for a cyanophagous polypeptide S-2L or one of its representative fragments involved in the transcription process, in particular a peptide of sequence SEQ ID N ° 92,143, 187,234 or one of their representative fragments.
  • the invention relates to a nucleotide sequence according to the invention characterized in that it codes for a cyanophagous polypeptide S-2L or one of its representative fragments involved in the viral virulence process, in particular a peptide of sequence SEQ ID No. 257 or a representative fragment.
  • the invention relates to a nucleotide sequence according to the invention characterized in that it codes for a cyanophagous polypeptide S-2L or one of its representative fragments involved in the functions relating to transposons in particular a peptide of sequence SEQ ID N ° 208 or one of its representative fragments.
  • the representative fragments of nucleotide sequences according to the invention can also be probes or primers, which can be used in methods of detection, identification, assay or amplification of nucleic sequences.
  • a probe or primer is defined, within the meaning of the invention, as being a fragment of single-stranded nucleic acids or a denatured double-stranded fragment comprising for example from 12 bases to a few kb, in particular from 15 to a few hundred bases, preferably from 15 to 50 or 100 bases, and having a specificity of hybridization under determined conditions to form a hybridization complex with a target nucleic acid.
  • the probes and primers according to the invention can be labeled directly or indirectly with a radioactive or non-radioactive compound by methods well known to those skilled in the art, in order to obtain a detectable and / or quantifiable signal.
  • the unlabeled polynucleotide sequences according to the invention can be used directly as a probe or primer.
  • sequences are generally marked to obtain sequences which can be used for numerous applications.
  • the labeling of the primers or probes according to the invention is carried out with radioactive elements or with non-radioactive molecules.
  • Non-radioactive entities are selected from ligands such as biotin, avidin, streptavidin, dioxygenin, haptens, dyes, luminescent agents such as radioluminescent, chemoluminescent, bioluminescent, fluorescent, phosphorescent agents.
  • the polynucleotides according to the invention can thus be used as a primer and / or probe in methods using in particular the PCR technique (polymerase chain reaction) (Rolfs et al., 1991, Berlin: Springer-Nerlag).
  • This technique requires the choice of pairs of oligonucleotide primers framing the fragment which must be amplified.
  • the amplified fragments can be identified, for example after agarose or polyacrylamide gel electrophoresis, or after a chromatographic technique such as gel filtration or ion exchange chromatography, and then sequenced.
  • the specificity of the amplification can be controlled by using, as primer, the nucleotide sequences of polynucleotides of the invention as template, plasmids containing these sequences or else the derived amplification products.
  • the amplified nucleotide fragments can be used as reagents in hybridization reactions in order to demonstrate the presence, in a biological sample, of a target nucleic acid of sequence complementary to that of said amplified nucleotide fragments.
  • the invention also relates to the nucleic acids capable of being obtained by amplification using primers according to the invention.
  • Other techniques for amplifying the target nucleic acid can advantageously be used as an alternative to PCR (PCR-like) using pairs of primers of nucleotide sequences according to the invention.
  • PCR-like we mean designate all the methods using direct or indirect reproductions of the nucleic acid sequences, or in which the labeling systems have been amplified, these techniques are of course known, in general it is the amplification of the DNA by a polymerase; when the original sample is an RNA, a reverse transcription should be carried out beforehand.
  • the target polynucleotide to be detected is an mRNA
  • an enzyme of reverse transcriptase type 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 method according to the invention.
  • the probe hybridization technique can be carried out in various ways (Matthews et al, 1988, Anal. Biochem., 169, 1-25).
  • the most general method consists in immobilizing the nucleic acid extracted from cells of different tissues or cells in culture on a support (such as nitrocellulose, nylon, polystyrene) and incubating, under well defined conditions, the target nucleic acid immobilized with the probe. After hybridization, the excess probe is eliminated and the hybrid molecules formed are detected by an appropriate method (measurement of radioactivity, fluorescence or enzymatic activity linked to the probe).
  • the latter can be used as capture probes.
  • a probe called a “capture probe”
  • a probe is immobilized on a support and is used 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.
  • a second probe called a “detection probe”, marked by an easily detectable element.
  • the antisense oligonucleotides that is to say those whose structure ensures, by hybridization with the target sequence, an inhibition of the expression of the corresponding product. Mention should also be made of sense oligonucleotides which, by interaction with proteins involved in the regulation of the expression of the corresponding product, will induce either an inhibition or an activation of this expression.
  • the probes or primers according to the invention are immobilized on a support, covalently or non-covalently.
  • the support can be a DNA chip or a high density filter, also objects of the present invention.
  • the term “DNA chip or high density filter” is intended to denote a support on which DNA sequences are fixed, each of which can be identified by its geographic location. These chips or filters differ mainly in their size, the material of the support, and possibly the number of DNA sequences attached to them.
  • the probes or primers according to the present invention can be fixed on solid supports, in particular DNA chips, by various manufacturing methods. In particular, a synthesis can be carried out in situ by photochemical addressing or by ink jet. Other techniques consist in carrying out an ex situ synthesis and in fixing the probes on the support of the DNA chip by mechanical, electronic or inkjet addressing. These different methods are known to those skilled in the art.
  • a nucleotide sequence (probe or primer) according to the invention therefore allows detection and / or amplification of specific nucleic acid sequences.
  • the detection of these said sequences is facilitated when the probe is fixed to a DNA chip, or to a high density filter.
  • DNA chips or high density filters indeed makes it possible to determine the expression of genes in an organism having a genomic sequence close to the cyanophagus S-2L.
  • the genomic sequence of cyanophagus S-2L serves as the basis for the construction of these DNA chips or filter.
  • the preparation of these filters or chips consists in synthesizing oligonucleotides, corresponding to the 5 'and 3' ends of the genes. These oligonucleotides are chosen using the genomic sequence and its annotations disclosed by the present invention.
  • the pairing temperature of these oligonucleotides at the corresponding places on the DNA must be approximately the same for each oligonucleotide. This makes it possible to prepare DNA fragments corresponding to each gene by the use of appropriate PCR conditions in a highly automated environment.
  • the amplified fragments are then immobilized on filters or supports in glass, silicon or synthetic polymers and these media are used for hybridization.
  • filters and / or chips and of the corresponding annotated genomic sequence makes it possible to study the expression of large sets, or even of all the genes in viruses close to the cyanophagus S-2L, by preparing the complementary DNAs, and by hybridizing them to DNA or to oligonucleotides immobilized on filters or chips. Also, the filters and / or the chips make it possible to study the variability of the strains by preparing the DNA of these viruses and by hybridizing them to the DNA or to the oligonucleotides immobilized on the filters or the chips.
  • the differences between the genomic sequences of the different strains or species can greatly affect the intensity of hybridization and, therefore, disturb the interpretation of the results. It may therefore be necessary to have the precise sequence of the genes of the strain that one wishes to study.
  • the use of high density filters and / or microchips provides new knowledge on the regulation of genes in organisms of industrial importance, and in particular recombinant bacteria incorporating genes from cyanophagus S-2L propagated under various conditions. It also allows rapid identification of the differences between the genomes of the strains used in multiple industrial applications.
  • DNA chips or filters according to the invention containing probes or primers specific for cyanophagus S-2L, are very advantageous elements of kits or necessary for the detection and / or quantification of the expression of genes of cyanophagus S-2L in recombinant bacteria integrating these genes.
  • the control of gene expression is a critical point for the metabolic pathways of cyanophagus S-2L, either by allowing the expression of one or more new genes, or by modifying the expression of genes already present in the cell.
  • the present invention provides all the naturally active sequences in cyanophagus S-2L allowing gene expression. It thus allows the determination of all the sequences expressed in cyanophagus S-2L. It also provides a tool for identifying genes whose expression follows a given pattern. To achieve this, the DNA of all or part of the genes of cyanophagus S-2L can be amplified using primers according to the invention, then fixed to a support such as for example glass or nylon or a DNA chip, in order to build a tool to monitor the expression profile of these genes.
  • This tool consisting of this support containing the coding sequences, serves as a hybridization matrix for a mixture of labeled molecules reflecting the messenger RNAs expressed in the cell (in particular the labeled probes according to the invention).
  • each control sequence present upstream of the segments serving as probes and to monitor their activity using an appropriate means such as a reporter gene (luciferase, ⁇ -galactosidase, GFP).
  • a reporter gene luciferase, ⁇ -galactosidase, GFP.
  • These isolated sequences can then be modified and assembled by metabolic engineering with sequences of interest with a view to their optimal expression.
  • the present invention gives the list of genes coding or capable of coding for proteins regulating the transcription of the genes of cyanophagus S-2L. Altering the structure or integrity of these genes could allow the expression of target genes controlled by target promoters of these regulators to be modified.
  • the indications given also allow those skilled in the art to choose the regulator or regulators relevant to the desired application as well as their target, which allows the optimization of the expression of genes of interest.
  • the invention relates to polypeptides comprising: a) a polypeptide coded by a nucleotide sequence according to the invention as defined above, in particular a polypeptide coded by an ORF; b) a polypeptide having at least 80%, preferably 85%, 90%, 95
  • the invention preferably relates to: a) the polypeptides of cyanophagus S-2L of sequences SEQ ID No. 2 to SEQ ID No. 527, coded respectively by ORFs 2 to 527, b) the 54 polypeptides mentioned in Table 1 ( SEQ ID N ° 14,18,26,68,86,92,105,109,134,142,143,148,152,169,175,187,
  • the invention also relates to: d) polypeptides having at least 80%, preferably 85%, 90%, 95% and
  • the invention obviously relates very especially to the polypeptides involved in the biosynthesis of D bases and metabolic intermediates of this biosynthesis, in particular the peptide of sequence SEQ ID No. 175 with succidinylate synthetase activity.
  • the phages for which the modifications are pre-replicative which is probably the case of cyanophages S-2L, have the protein coding sequences necessary for the biosynthesis of the modified bases, in the present case of the D bases.
  • the polymerase enzymes in particular DNA polymerases, must be able to have base D specifically as a substrate instead of base A.
  • the DNA polymerase of cyanophagus S-2L is thus capable of discriminate dDTP from dATP. Likewise, transcription depends on a specific RNA polymerase and / or on a specific sigma factor.
  • the invention therefore relates, according to a preferred embodiment, to the specific polypeptides with DNA polymerase, RNA polymerase activity and associated factors, in particular the peptides of sequence SEQ ID No. 92 and SEQ ID No. 234 which have specific activities of transcription of DNA comprising bases D.
  • polypeptides polypeptide sequences, peptides and proteins are interchangeable. It should be understood that the invention does not relate to polypeptides in natural form, that is to say that they are not taken in their natural environment but that they could have been isolated or obtained by purification from natural sources, or obtained by genetic recombination, or by chemical synthesis, and that they can then contain non-natural amino acids as will be described later.
  • polypeptide having a certain percentage of identity with another which will also be designated by homologous polypeptide, is meant to designate the polypeptides exhibiting, relative to the natural polypeptides, certain modifications, in particular a deletion, addition or substitution of at least one amino acid, a truncation, an elongation, a chimeric solution and / or a mutation, or the polypeptides exhibiting post- translational.
  • homologous polypeptides those whose amino acid sequence have at least 80%, preferably 85%, 90%, 95% and 98% of homology with the amino acid sequences of the polypeptides according to the invention are preferred. .
  • equivalent amino acids aims here to designate any amino acid capable of being substituted for one of the amino acids of the basic structure without however essentially modifying the biological activities of the corresponding peptides and as they will be defined by the following.
  • Leucine can thus be replaced by valine or isoleucine, aspartic acid by glutamine acid, glutamine by asparagine, arginine by lysine, etc. reverse substitutions being naturally possible in the same conditions.
  • the homologous polypeptides also correspond to the polypeptides encoded by the homologous or identical nucleotide sequences, as defined above and thus include, in the present definition, polypeptides which are mutated or correspond to inter or intra species variations, which may exist in the cyanophagus S-2L, and which correspond in particular to truncations, substitutions, deletions and / or additions, of at least one amino acid residue. It is understood that the percentage of identity between two polypeptides is calculated in the same way as between two nucleic acid sequences. Thus, the percentage of identity between two polypeptides is calculated after alignment optimal of these two sequences, on a window of maximum homology. To define said maximum homology window, the same algorithms can be used as for the nucleic acid sequences.
  • biologically active fragment of a polypeptide according to the invention is intended to denote in particular a fragment of polypeptide comprising at least 5 amino acids, preferably at least 7, 10, 15, 25, 50, 75, 100,150, 200, 250 , 300 amino acids, having at least one of the biological characteristics of the polypeptides according to the invention, in particular in that it is capable of generally exercising even partial activity, such as for example: - enzymatic (metabolic) activity or an activity which may be involved in the biosynthesis or biodegradation of organic or inorganic compounds;
  • Polypeptide fragments can also be prepared by chemical synthesis, from hosts transformed by an expression vector according to the invention which contain a nucleic acid allowing the expression of said fragment, and placed under the control of regulatory elements and / or appropriate expression.
  • modified polypeptide of a polypeptide according to the invention is intended to denote a polypeptide obtained by genetic recombination or by chemical synthesis as described below, which exhibits at least one modification with respect to the normal sequence. These modifications can be carried in particular on amino acids necessary for the specificity or the efficiency of the activity, or at the origin of the structural conformation, of the charge, or of the hydrophobicity of the polypeptide according to the invention. It is thus possible to create polypeptides of equivalent, increased or decreased activity, or of equivalent specificity, narrower or wider.
  • modified polypeptides mention should be made of the polypeptides in which up to five amino acids can be modified, truncated at the N or C-terminus, or else deleted, or added.
  • modifications of a polypeptide are aimed in particular: - to allow its implementation in processes of biosynthesis or biodegradation of organic or inorganic compounds,
  • Chemical synthesis also has the advantage of being able to use unnatural amino acids or non-peptide bonds. Thus, it may be advantageous to use unnatural amino acids, for example in D form, or analogs of amino acids, in particular suffering forms.
  • the invention relates to a polypeptide according to the invention, characterized in that it is a cyanophagous polypeptide S-2L or one of its representative fragments involved in the metabolism of nucleotides, purines, pyrimidines or nucleosides.
  • the invention relates to a polypeptide according to the invention, characterized in that it is a cyanophagous polypeptide S-2L or one of its representative fragments involved in the replication process, and in that it is chosen from the polypeptides of sequence SEQ ID No. 14,18,142,355,429,454 and one of their fragments.
  • the invention very advantageously relates to cyanophagous S2L polypeptides of at least 7 amino acids and having adenylosuccinate activity synthetase.
  • such fragments comprise the motif GSTGKG.
  • the inventors have indeed identified consensus sites, in particular the phosphate and IMP binding site zones. .
  • the invention relates to a polypeptide according to the invention, characterized in that it is a cyanophagous polypeptide S-2L or one of its fragments involved in the process of transcription, and in that it is chosen from the polypeptides of sequence SEQ ID No. 92,143,187 and one of their representative fragments.
  • the invention relates to a polypeptide according to the invention, characterized in that it is a cyanophagous envelope polypeptide S-2L or one of its fragments, and in that it is chosen from the polypeptides corresponding to ORF169,316,351,392,395,406,422,425 and one of their representative fragments.
  • the invention relates to a polypeptide according to the invention, characterized in that it is a cyanophagous polypeptide S-2L or one of its representative fragments involved in the hijacking of cellular machinery or in intermediate metabolism.
  • the invention relates to a polypeptide according to the invention, characterized in that it is a cyanophagous polypeptide S-2L or one of its representative fragments involved in the virulence process, in particular the polypeptide of sequence SEQ ID No. 247 and one of its representative fragments.
  • the invention relates to a polypeptide according to the invention, characterized in that it is a polypeptide of cyanophagus S-2L or one of its fragments involved in the functions relating to transposons, in particular the polypeptide of sequence SEQ ID No. 208 and one of its representative fragments.
  • a subject of the present invention is also the nucleotide and / or polypeptide sequences according to the invention, characterized in that said sequences are recorded on a recording medium the shape and nature of which facilitate reading, analysis and / or the exploitation of said sequence (s).
  • These supports can also contain other information extracted from the present invention, in particular analogies with already known sequences, as mentioned in Table 1 and / or information concerning the nucleotide sequences and / or polypeptides of other microorganisms in order to facilitate comparative analysis and use of the results obtained.
  • recording media particular preference is given to media readable by a computer, such as magnetic, optical, electrical or hybrid media, in particular computer floppy disks, CD-ROMs, computer servers. Such recording media are also subject of the invention.
  • the recording media according to the invention are very useful for the choice of primers or nucleotide probes for the determination of genes in the cyanophagus S-2L or strains close to this organism.
  • the use of these supports for the study of the genetic polymorphism of strain close to the cyanophagus S-2L, in particular by the determination of the regions of collinearity is very useful insofar as these supports provide not only the sequence nucleotide of the cyanophagous S-2L genome, but also the genomic organization in said sequence.
  • the uses of recording media according to the invention are also objects of the invention.
  • a method of studying the genetic polymorphism between strains close to the cyanophagus S-2L, by determining the regions of collinearity can comprise the steps of
  • sequence comparison software such as the Blast software, or the software of the GCG kit, described above.
  • the invention also relates to a nucleotide sequence as described above, immobilized on a support, covalently or non-covalently, in particular a high density filter or a DNA chip.
  • the invention also relates to a nucleotide sequence as described above for the detection and / or amplification of nucleic sequences.
  • such a detection and amplification method comprises for example the following steps: a) optionally, isolation of the DNA from the biological sample to be analyzed, or obtaining a cDNA from the RNA biological sample; b) specific amplification of the DNA of cyanophages S-2L using at least one primer according to the invention; c) highlighting of the amplification products.
  • This process is based on specific amplification of DNA, in particular by an amplification chain reaction.
  • a method is also preferred comprising the following steps: a) bringing a nucleotide probe according to the invention into contact with a biological sample, the nucleic acid contained in the biological sample having, if necessary, previously been made accessible to hybridization, under conditions allowing hybridization of the probe to the nucleic acid of cyanophagus S-2L; b) demonstration of the hybrid possibly formed between the nucleotide probe and the DNA of the biological sample.
  • a method should not be limited to the detection of the presence of the DNA contained in the certified biological sample, it can also be implemented to detect the RNA contained in said sample. This process includes in particular the Southern and Northern blot.
  • the present invention also includes a kit or kit for the detection and / or identification of cyanophagus S-2L, characterized in that it comprises the following elements: a) a nucleotide probe according to the invention; b) optionally, the reagents necessary for carrying out a hybridization reaction; c) optionally, at least one primer according to the invention as well as the reagents necessary for a DNA amplification reaction.
  • kits or kits for the detection and / or identification of cyanophagus S-2L comprising the following elements: a) a nucleotide probe, called capture probe, according to the invention; b) an oligonucleotide probe, called the revelation probe, according to the invention; c) optionally, at least one primer according to the invention as well as the reagents necessary for a DNA amplification reaction.
  • the invention also relates to the cloning and / or expression vectors, which contain a nucleotide sequence according to the invention. Particularly preferred are the nucleotide sequences encoding polypeptides involved in nucleotide metabolism, purines, pyrimidines or nucleosides.
  • the vectors according to the invention preferably comprise elements which allow the expression and / or the secretion of the nucleotide sequences in a determined host cell.
  • the vector must then include a promoter, initiation signals and translation termination, as well as appropriate regions for transcription regulation. It must be able to be maintained stably in the host cell and may possibly have specific signals which specify the secretion of the translated protein. These various elements are chosen and optimized by a person skilled in the art according to the cell host used. To this end, the nucleotide sequences according to the invention can be inserted into vectors with autonomous replication within the chosen host, or can be vectors integrating with the chosen host.
  • Such vectors are prepared by methods commonly used by those skilled in the art, and the resulting clones can be introduced into an appropriate host by standard methods, such as lipofection, electroporation, heat shock, or chemical methods .
  • the vectors according to the invention are for example vectors of plasmid or viral origin. They are useful for transforming host cells in order to clone or express the nucleotide sequences according to the invention.
  • the cyanophagus S-2L itself can be used directly as a vector.
  • the invention also includes host cells transformed with a vector according to the invention.
  • the cell host can be chosen from prokaryotic or eukaryotic systems, for example bacterial cells but also yeast cells or animal cells, in particular mammalian cells. You can also use insect cells or plant cells.
  • the preferred host cells according to the invention are in particular prokaryotic cells.
  • the cells transformed according to the invention can be used in processes for the preparation of recombinant polypeptides according to the invention.
  • the processes for preparing a polypeptide of interest according to the invention in recombinant form, outside the natural environment, characterized in that they use a vector and / or a cell transformed with a vector according to the invention are themselves included in the present invention.
  • the use of cyanophagus S-2L for the production of such peptides therefore also forms part of the invention.
  • a cell transformed with a vector according to the invention is cultivated under conditions which allow the expression of said polypeptide. of interest and said recombinant peptide is recovered.
  • the host cells according to the invention can also be used for the preparation of food compositions, which are themselves subject of the present invention.
  • Such a process for obtaining proteins of interest from the cyanophagus S-2L comprises the insertion of genes of interest from the genome of the phage S-2L, typically by ligation, into cloning and expression vectors , under conditions allowing their expression by the taking over by the replication machinery of a host organism such as E. coli, and the extraction of the proteins produced.
  • Hereditary folding messages copied in the form of canonical DNA are able to express themselves as cyanobacteria genes.
  • the messenger RNAs issued after rewriting S-2L DNA in E. coli are translated into proteins identical to those produced during infection of Synechococcus with S-2L.
  • polypeptides of interest are proteins involved in the metabolism of D bases, in particular succinyladenylate synthetase.
  • D base is probably formed by pre-replicative modification and that cellular genes have been recruited for this purpose, two biosynthetic pathways appearing to form dDTP from a deoxynucleotide. canonical, either dAMP or dGMP.
  • the activated monomer dATP is immediately hydrolyzed into dAMP by an enzyme of the type coded by dut in E. coli (9) or of the product of the mutT (9) gene, which has has the double effect of blocking the access of dATP to DNA synthesis and of providing the precursor of DMP. Its biosynthesis takes place according to the two successive reactions converting IMP to GMP in cellular metabolism (9); the nucleotide is finally activated in dDTP in two stages of phosphorylation.
  • dDMP is obtained by applying to dGMP the two reactions converting in IMP cells to AMP (9). If it also takes dATP as a precursor, this second path is longer because dGMP must first be synthesized in dIMP life. Throughout this second pathway, three specific and mutagenic dNTPs are formed (dIMP, dXMP and dSMP), against a single (diGMP) in the first ( Figure 2a).
  • the polypeptides of interest are cyanophagous polymerases S-2L, capable of polymerizing D bases, which allows the propagation of nucleic acids incorporating D bases in vitro and in vivo.
  • DNA polymerases specialized in high stability duplexes which are incapable of replicating dA taken as a constituent of the matrix or as a triphosphate monomer.
  • These DNA polymerases will typically be obtained by a process comprising a step of expression, outside the natural environment, of the gene for said DNA polymerase in recombinant bacteria.
  • the polypeptides of interest are polypeptides capable of modifying the transcription of the DNA of host cells of the cyanophagus S-2L.
  • T4 alter the RNA polymerase of E. coli.
  • S-2L deviates from the consensus known to those skilled in the art (TATA box in particular). It is likely that transcription initiation factors (sigma or other) are coded or modified by the phage, or even that they are embedded in the capsid to allow the initiation of the viral program. Be that as it may, the sequencing carried out by the inventors makes it possible to identify without excessive effort certain genes of S-2L responsible for the control of transcription by chemical alteration of the DNA.
  • the cell host can be chosen from prokaryotic or eukaryotic systems.
  • a vector according to the invention carrying such a sequence can therefore be advantageously used for the production of recombinant proteins, intended to be secreted. Indeed, the purification of these recombinant proteins of interest will be facilitated by the fact that they are present in the supernatant of the cell culture rather than inside the host cells.
  • the polypeptides according to the invention can also be prepared by chemical synthesis. Such a preparation process is also an object of the invention.
  • a person skilled in the art knows the chemical synthesis processes, for example the techniques implementing solid phases (see in particular Steward et al., 1984, Solid phase peptides synthesis, Pierce Chem. Company, Rockford, 111, 2nd ed., (1984)) or techniques using partial solid phases, by condensation of fragments or by synthesis in conventional solution.
  • the polypeptides obtained by chemical synthesis and which may contain corresponding unnatural amino acids are also included in the invention.
  • the invention also includes the hybrid polypeptides which comprise at least the sequence of a polypeptide according to the invention, and the sequence of a polypeptide capable of inducing an immune response in humans or animals.
  • the invention also includes the nucleotide sequences which code for such hybrid polypeptides, or the vectors which contain these nucleotide sequences.
  • This coupling between a polypeptide according to the invention and an immunogenic polypeptide of interest can be carried out chemically, or biologically.
  • the bifunctional reagents allowing this coupling are determined as a function of the end chosen to achieve this coupling, and the coupling techniques are well known to those skilled in the art.
  • the conjugates resulting from a coupling of peptides can also be prepared by genetic recombination.
  • the hybrid (conjugated) peptide can in fact be produced by recombinant DNA techniques, by insertion or addition to the DNA sequence coding for the polypeptide according to the invention, of a sequence coding for the peptide (s) ) antigen (s), immunogenogen (s) or hapten (s).
  • said immune polypeptide is chosen from the group of peptides containing toxoids, in particular the diphtheria toxoid or the tetanus toxoid, proteins derived from Streptococcus (such as the protein for binding to human seralbumin), OMPA membrane proteins and complexes. proteins from external membranes, vesicles from external membranes or thermal shock proteins.
  • nucleotide and vector sequences coding for a hybrid polypeptide according to the invention are also subject of the invention.
  • hybrid polypeptides according to the invention are very useful for obtaining monoclonal or polyclonal antibodies capable of specifically recognizing the polypeptides according to the invention. Indeed, a hybrid polypeptide according to the invention allows the potentiation of the immune response, against the polypeptide according to the invention coupled to the immunogenic molecule. Such monoclonal or polyclonal antibodies, their fragments, or chimeric antibodies, recognizing the polypeptides according to the invention, are also objects of the invention.
  • the specific monoclonal antibodies can be obtained according to the conventional method of hybridoma culture described by Kohler and Milstein (1975, Nature 256, 495).
  • the antibodies according to the invention are, for example, chimeric antibodies, humanized antibodies, Fab fragments, or F (ab ') 2 . It can also be in the form of an immunoconjugate or of labeled antibodies in order to obtain a detectable and / or quantifiable signal.
  • the antibodies according to the present invention are in particular usable in order to detect an expression of a cyanophagous gene S-2L.
  • the presence of the expression product of a gene recognized by an antibody specific for said expression product can be detected by the presence of an antigen-antibody complex formed after contacting a recombinant bacterium expressing a d gene. given interest of cyanophagus S-2L with an antibody according to the invention.
  • the bacterial strain used may have been "prepared", that is to say centrifuged, lysed, placed in a reagent suitable for constituting the medium suitable for the immunological reaction.
  • a method of detecting the expression of a gene, corresponding to a Western blot which can be carried out after a polyacrylamide gel electrophoresis of a lysate of the bacterial strain, in the presence or in the absence of reducing conditions (SDS-PAGE). After migration and separation of the proteins on the polyacrylamide gel, said proteins are transferred to an appropriate membrane (for example made of nylon) and the presence of the protein or polypeptide of interest is detected, by bringing said membrane into contact with a antibody according to the invention.
  • an appropriate membrane for example made of nylon
  • polypeptides and antibodies according to the invention can advantageously be immobilized on a support, in particular a protein chip.
  • a protein chip is an object of the invention, and may also contain at least one polypeptide from a microorganism other than cyanophagus S-2L or an antibody directed against a compound of a microorganism other than cyanophagus S-2L.
  • the protein chips or high density filters containing proteins according to the invention can be constructed in the same way as the DNA chips according to the invention.
  • the latter method is preferable, when it is desired to attach proteins of large size to the support, which are advantageously prepared by genetic engineering.
  • an antibody according to the invention is fixed on the support of the protein chip, and the presence of the corresponding antigen, specific for Cyanophage S-2L or an associated microorganism, is detected.
  • a protein chip described above can be used for the detection of gene products, to establish an expression profile of said genes, in addition to a DNA chip according to the invention.
  • the protein chips according to the invention are also extremely useful for proteomics experiments, which studies the interactions between the different proteins of a given microorganism.
  • proteomics experiments which studies the interactions between the different proteins of a given microorganism.
  • peptides representative of the various proteins of an organism are fixed on a support. Then, said support is brought into contact with labeled proteins, and after an optional rinsing step, interactions between said labeled proteins and the peptides attached to the protein chip.
  • protein chips comprising a polypeptide sequence according to the invention or an antibody according to the invention are subject of the invention, as well as the kits or kits containing them.
  • the primers and / or probes and / or polypeptides and / or antibodies according to the present invention used in the methods according to the present invention are chosen from primers and / or probes and / or polypeptides and / or antibodies specific for Cyanophage S -2L
  • the present invention also relates to the strains of Cyanophage S-2L and / or associated microorganisms containing one or more mutation (s) in a nucleotide sequence according to the invention, in particular an ORF sequence, or their regulatory elements (in particular promoters).
  • strains of Cyanophage S-2L having one or more mutation (s) in the nucleotide sequences coding for polypeptides involved in the metabolism of D bases, replication and transcription are preferred, according to the present invention.
  • Said mutations can lead to inactivation of the gene, or in particular when they are located in the regulatory elements of said gene, to overexpression of the latter.
  • strains of Cyanophage S-2L overexpressing a polypeptide according to the invention involved in the functions relating to the synthesis of bases D or of polynucleotides incorporating at least one base D.
  • the invention also relates to the use of the polypeptide sequences as described above for the production of bases D and or of polynucleotide sequences comprising bases D.
  • These polynucleotide sequences will in particular be DNA or RNA sequences, in particular mRNA.
  • the invention relates to a method for obtaining bases D and / or polynucleotides of interest comprising at least one base D, said method comprising the culture of a microorganism containing at least one nucleotide sequence of cyanophagus S- 2L coding for at least one polypeptide involved in the synthesis of bases D, under conditions suitable for the development of the vector and the synthesis of bases D.
  • the cultivated microorganism comprises a vector as described previously containing the said nucleotide sequence (s) of cyanophagus S-2L coding.
  • such a method comprises:
  • such a method comprises:
  • restriction enzymes whose restriction sites do not have an A base, in particular Smal (site CCCGGG), SacII (site CCGCGG), Mspl (CCGG site), BspRI (GGCC site).
  • restriction enzymes comprising at least one base A do not hydrolyze the DNA of S-2L: BamHI (GCATCC), EcoRI (GAATTC), HindIII (AAGCTT), Sau3AI (GATC).
  • the invention relates to a process for obtaining bases D and / or polynucleotides of interest comprising at least one base D, said process comprising:
  • the invention relates to a process for obtaining polynucleotides of interest comprising at least one base D, said process comprising the culture of a microorganism containing at least one nucleotide sequence of cyanophagus S-2L coding for at least one polypeptide involved in the elongation of said polynucleotides with incorporation of bases D, DNA polymerase in particular, under conditions suitable for the development of the vector and the elongation of said polynucleotides.
  • the invention relates to the use of the cyanophagus S-2L for the production of reagents useful for PCR or PCR Like reactions involving D bases.
  • these reagents will be dDTP monomers.
  • the dDTP monomer is a good substrate for the DNA polymerases of cyanophagus S-2L, and matrices comprising the base D are effectively replicated (1).
  • the biotechnological production of dD, dDMP and dDTP thus applies to PCR techniques, by increasing the thermal stability of duplexes, or by masking and unmasking numerous restriction sites (10). It is understood that this production is not production in the natural environment, production in the natural environment signifying production by the cyanophagus S-2L itself.
  • the invention also relates to a method for producing polynucleotides of interest comprising at least one base D, said method comprising a step of amplification, in the presence of cyanophagous polymerase D and suitable primers, of polynucleotides comprising at least one base D. Thanks to this method, according to a technique of PCR or PCR like type, from a polynucleotide of interest comprising at least one base D of known sequence, a large number of copies of this nucleotide are obtained.
  • the gene involved in the synthesis of polynucleotides of interest comprising at least one base D is the gene for succinyladenylate synthetase.
  • succinyladenylate synthetase catalyzes the reaction of dGMP to dSMP itself transformed into dDMP ( Figure 2).
  • the polynucleotides of interest are nucleosides of therapeutic interest.
  • the polynucleotides of interest are produced by hemisynthesis or by fermentation.
  • the invention further relates to a method for selecting compounds capable of stimulating or inhibiting the synthesis of bases D and / or of polynucleotides of interest incorporating at least one base D, comprising the addition to the synthesis medium of the compound tested and comparing the synthesis in the presence and absence of said compound.
  • the invention relates to the use of the cyanophagous nucleotide sequences S-2L as described above for testing their function in the metabolism of nucleotides, purines, pyrimidines or nucleosides, replication and transcription.
  • the invention relates to the use of the cyanophagus S-2L for the determination of genes making it possible to repair the G: T or iG: T mismatches occurring by deamination.
  • base D itself is known as a mutagen in E. coli.
  • iG isoguanine
  • M. Bouzon, P. Marlière, unpublished results isoguanine
  • Deamination of D at position 6 leads to guanine. That this last deamination reaction occurs after incorporation of D into DNA, it will result in a mutation in the next replication cycle. Thanks to the sequencing performed, the identification of genes capable of repairing G: T or iG: T mismatches occurring by deamination is now possible.
  • the invention relates to the use of the cyanophagus S-2L for the identification of genes and the production of proteins capable of regenerating 5'-termini.
  • the replication of cyanophagous DNA whose stability is high (7,8), could moreover require tailor-made auxiliary proteins (helicase, S SB).
  • the genome is made up of a linear duplex, which supposes a 5'-termini regeneration machinery, like the endonuclease used to resolve concatemers in T7 (4), or the 5 'adduction protein in phi29 ( 6), whose activity may require the presence of D in their substrates.
  • the invention relates to the use of the cyanophagus S-2L for the identification of genes capable of modulating the activity of ribosomes
  • the cyanophagus S-2L is moreover capable of forming a ribonucleotide precursor carrying the base D, to then reduce it to a corresponding deoxyribonucleotide, as happens with the four bases of the RNA (9).
  • the transcription and translation of the phage genes could be carried out via the use of codons, or even tRNAs as in T4 and T5, comprising this base. If such an option was taken by the phage, it is possible that some of its genes modulate the activity of ribosomes.
  • the invention relates to the use of the cyanophagus S-2L for the identification or the production of compounds which inhibit the biosynthesis of purine nucleotides.
  • phage genomes specify a whole range of inhibitors targeting cellular enzymes such as thymidylate synthase, dUTPase, etc. (11).
  • S-2L the inventors can now identify inhibitors capable of affecting the biosynthesis of purine nucleotides.
  • the invention thus also relates to a method using such inhibitors to control the metabolism or the genetic expression of cells capable of being infected by a cyanophagus S2L, in particular cyanobacteria.
  • the invention also relates to a method using such inhibitors to control this metabolism.
  • Figures 2a and 2b represent two possible biosynthetic pathways for the synthesis of bases D by the cyanophagus S-2L, the path of Figure 2b being the most likely
  • FIG. 4 schematically represents the potential difficulty of cloning genes incorporating D bases in E.Coli.
  • S2L cyanophages are cultivated in mass from the species
  • Synechococcus elongatus (8) The extracted DNA is fragmented by sonication to constitute a shotgun library cloned into a vector in E. coli. The clones are sequenced intensively on a sequencer until the genome is completely covered.
  • ORFs are elucidated as homologs to known genes, their expression is carried out in E. coli or in Synechococcus, according to the functions assumed, in particular with the aim of validating the functional hypotheses or exploring the synthetic potentials.
  • FIG. 2 the supposed intermediates of the synthesis pathways (FIG. 2) have been synthesized according to current methods of the chemistry of nucleosides and nucleotides. They are systematically subjected to extracts or mixtures of extracts of recombinant strains each expressing an S-2L gene, in order to identify the enzymatic activities specified by the phage.
  • the DNA of phage S-2L was prepared from Synechoccus culture lysate. elongatus by adapting the techniques used to prepare the phage ⁇ DNA.
  • This DNA was digested with various restriction enzymes, including Smal, which made it possible to verify that the restriction profile obtained was identical to that described. Then, it was shown that S-2L DNA can be replicated in E. coli and sequenced according to standard protocols, which led to the construction of a total library.
  • This library was constructed by inserting DNA fragments digested by the enzyme CviJI (of size between 3 and 5 kb) in the plasmid pBAM digested by the enzyme SmaI and dephosphorylated. After electroporation of the strain of E.
  • tail protein MLKI and J proteins involved in the formation and assembly of the tail of the bacteriophage ⁇ : tail protein MLKI and J, of the protein GP17 which plays a role in the packaging of DNA in the bacteriophage T4, an exonuclease which could intervene in the exclusion of base A, an RNA helicase, a sigma factor and a succinyladenylate synthetase.
  • 2,6-diaminopurine becomes toxic (10 mM) for ⁇ . coli when it is in phosphorylated form (which has been tested in the same strain of col. coli expressing the yaaG gene of Bacillus subtilis or MG1655 pSU yaaG) which makes it possible to have a screen to identify the complete pathway in vivo of base D biosynthesis.
  • complete identification is not necessary to obtain D bases now by the methods described above.
  • Another approach consists in systematically expressing the ORFs specifying all the possible S-2L genes and in combining the crude activities resulting from this expression in order to make the metabolites of the pathway appear in vitro.
  • An inducible metabolic pathway producing dDTP will then be built in E. coli by assembling the appropriate genes. The path thus constructed will be applied to synthetic precursors to generate nucleotides deviating from the base and from sugar.
  • base D in the replication and transcription processes is systematically sought in extracts from bacteria expressing phage ORFs.
  • the above results were obtained using the following works.
  • the ddbA gene was expressed in E. coli under the control of an inducible promoter and several tests were carried out in order to determine the activity of the corresponding protein.
  • the results obtained show that the expression of ddbA makes it possible to restore the growth of E. coli in the presence of a high concentration of dGMP.
  • 2,6-diaminopurine becomes toxic for E. coli when it is in phosphorylated form which should make it possible to have a screen to identify in vivo the complete pathway for base D biosynthesis.
  • the ddbA gene was amplified using 100 pmol of each oligonucleotide ngaattcaagctttcagcgacggtagcgggcatac and nnnnccatggtgaagaactgcaacctgatc, lOOng of S-2L DNA as template DNA, 200mM of each dNTPs, 10 ml of Pfu polymeraseU buffer 10% concentrated 10-fold Pfu polymeraseu.
  • the amplification cycles were: a stage of 10 min at 95 ° C, then 25 cycles 95 ° C 30 sec, 56 ° C 30 sec, 72 ° C 2 min 20 sec then a step of 10 min at 72 ° C.
  • the amplification product was then purified using the Kit
  • the amplification product was inserted into the plasmid pBAD24 (Guzman et al., 1995 J Bacteriol 177: 4121-4130) digested with the same restriction enzymes.
  • the ddbA gene is in this construction expressed from the promoter of the araBAD operon which is inducible by arabinosis.
  • the cyanophagous bank S2L is maintained in the strain of E. Coli B2033 deposited on January 24, 2001 at the National Collection of Cultures of Microorganisms, Institut Pasteur, 25 rue du Dr Roux, 75724 PARIS Cedex 15, France, according to the provisions of Budapest Treaty, and was registered under serial number 1-2619.

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EP03747467A 2002-04-30 2003-04-28 Genbank von cyanophage s-2l und deren funktionsanalyse Withdrawn EP1499713A2 (de)

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FR0205424 2002-04-30
FR0205424A FR2839079B1 (fr) 2002-04-30 2002-04-30 Banque genomique du cyanophage s-2l et analyse fonctionnelle partielle
PCT/FR2003/001328 WO2003093461A2 (fr) 2002-04-30 2003-04-28 Banque genomique du cyanophage s-2l et analyse fonctionnelle

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WO2022214617A1 (en) 2021-04-07 2022-10-13 Institut Pasteur 2-aminoadenine modified nucleic acids, cells comprising them, and methods of producing them

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EP3378937B1 (de) * 2015-11-19 2021-06-09 Niigata University Peptid mit spezifischer akkumulation für bauchspeicheldrüsenkrebs und verwendung dieses peptids
EP4389216A3 (de) * 2017-06-19 2024-11-06 Allegro Pharmaceuticals, LLC Peptidzusammensetzungen und therapeutische verwendungen
US12454549B2 (en) 2017-06-19 2025-10-28 Jenius Pharma, Llc Treatments for diseases and disorders that involve oxidative stress
WO2022152192A1 (zh) * 2021-01-14 2022-07-21 天津大学 参与噬菌体二氨基嘌呤合成的酶及其应用
WO2022219033A1 (en) * 2021-04-15 2022-10-20 The European Syndicate Of Synthetic Scientists And Industrialists Novel family of dna polymerases accepting 2-aminoadenine and rejecting adenine in their substrates

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Title
ROST B.: "Enzyme function less conserved than anticipated", J. MOL. BIOL., vol. 318, 2002, pages 595 - 608, XP004449547, DOI: doi:10.1016/S0022-2836(02)00016-5 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022214617A1 (en) 2021-04-07 2022-10-13 Institut Pasteur 2-aminoadenine modified nucleic acids, cells comprising them, and methods of producing them

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US20060270005A1 (en) 2006-11-30
AU2003249159A8 (en) 2003-11-17
WO2003093461A2 (fr) 2003-11-13
FR2839079B1 (fr) 2007-10-12
FR2839079A1 (fr) 2003-10-31
WO2003093461A8 (fr) 2004-06-24
CA2483706A1 (fr) 2003-11-13
AU2003249159A1 (en) 2003-11-17

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