Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2217/00—Genetically modified animals
  • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)

Definitions

• the subject of the invention is the genome sequence of Pyrococcus abyssi, nucleotide sequences coding for polypeptides of Pyrococcus abyssi, such as polypeptides involved in the metabolism or in the replication process, as well as vectors including said sequences and cells transformed by these vectors.
• the invention also relates to methods using these nucleic acids or polypeptides, in particular methods of biosynthesis or biodegradation of molecules of interest, as well as kits comprising these polypeptides.
• thermococcals belonging to the branch of euryarchaeotes, and particularly the genus Pyrococcus, are of particular interest to clarify the phylogenetic relationships within archae and to address questions relating to the nature of the last universal ancestor. common to eukaryotes, bacteria and archaebacteria (LUC A: Last Universal Common Ancestor) and at the origin of life.
• Archaebacteria of the genus Pyrococcus are currently the preferred models for the study of hyperthermophilic organisms (optimal growth temperatures between 80 and 110 ° C) (Adams Annu. Rev. Microbiol., 47: 627, 1997). Although anaerobic, these organisms are easy to cultivate and can be obtained in the form of colonies on a dish (in Gelrite medium).
• the proteins of archaebacteria of the genus Pyrococcus are intrinsically thermostable, some of them being able to resist incubation for several tens of minutes at temperatures above 110 °.
• hyperthermophilic proteins can be used in numerous industrial processes such as, for example, DNA polymerases, DNA ligases, DNA modification enzymes, inteins, proteases or enzymes of sugar metabolism.
• DNA polymerases DNA ligases
• DNA modification enzymes DNA modification enzymes
• inteins DNA modification enzymes
• proteases enzymes of sugar metabolism.
• the stability of hyperthermophilic proteins also often facilitate their crystallization and the quality of the crystals obtained, which promotes their structural study.
• Pyrococcus furiosus the most studied species, which was isolated from a terrestrial hydrothermal source and whose genome, partially available, a was sequenced by Robert Weiss's team at the University of Utah.
• horikoshii genome contains only 20% of identifiable genes, which is low compared to the genomes of other archaebacteria such as Methanococcus jannashii, Archaeoglobus fiilgidus and Methanobacterium thermoautotrophicum, for which the proportion of genes identifiable by sequence similarity is around 40%.
• the bacteria of the genus Pyrococcus are therefore a reservoir of genes potentially of industrial interest, including many genes which are still unknown.
• pGT5 This plasmid, called pGT5, has been sequenced and identified as a rolling circle type plasmid (Erauso et al., 1996, J. Bacteriol. 178: 3232) and is currently used as a starting point for the development of a shuttle vector for Pyrococcus (Aagard et al., FEMS Microbiol. Rev. 18, 93, 1996; Aravalli et al., Extemophiles 1997, 1: 183-91). Due to the presence of the plasmid pGT5, P. abyssi was also chosen as the preferred strain for the study of DNA replication in hyperthermophilic archaebacteria (Marsin and Forterre, Mol. Microbiol., 27, 1183-1192, 1998).
• Pyrococcus of different species will in particular allow the identification of genes that can be used as genetic markers and use the power of comparative genomics to improve the identification of genes and their functional annotation (which is particularly important in the case of Pyrococcus given the large number of orphan genes), and provide important information about the plasticity of their genome.
• having three genomic sequences is particularly interesting because this will also allow to polarize the genomic events which occurred between two of them (the closest ones, P. abyssi and P. horikoshii) in using the third (P. furiosus) as the external group.
• the subject of the present invention is the nucleotide sequence of sequence SEQ ID No. 1 of the genome of Pyrococcus abyssi strain ORSAY.
• the ORSAY Pyrococcus abyssi strain was the subject of a deposit on April 9, 1999, at the CNCM (Paris, France) under the number 1-2180.
• the present invention also relates to nucleotide sequences characterized in that they are chosen from: a) a nucleotide sequence comprising at least 99.9% identity with the sequence SEQ ID No. 1; b) a nucleotide sequence homologous to the sequence SEQ ID No. 1; c) a nucleotide sequence complementary to the sequence SEQ ID No.
• nucleotide sequence 1 or complementary to a nucleotide sequence as defined in a), or b), and a nucleotide sequence of their corresponding RNA; d) a nucleotide sequence of a fragment representative of the sequence SEQ ID No. 1, or of a fragment representative of the nucleotide sequence as defined in a), b) or c); e) a nucleotide sequence comprising a sequence as defined in d); f) a nucleotide sequence capable of being obtained from a nucleotide sequence as defined in a), b), c), d) or e); and g) a nucleotide sequence modified from a nucleotide sequence as defined in a), b), c), d), e) or 0-
• genomic sequence of Pyrococcus abyssi is meant to denote the chromosome sequence of Pyrococcus abyssi.
• nucleotide sequence, polynucleotide or nucleic acid is understood to mean both double-stranded DNA, single-stranded DNA and transcripts of said DNAs.
• the present invention does not relate to the genomic nucleotide sequences of Pyrococcus abyssi taken in their natural environment, that is to say in the natural state.
• sequences which could have been isolated, purified or partially purified, using separation methods such as, for example, ion exchange chromatography, exclusion based on molecular size, or affinity, or else fractionation techniques based on the solubility in different solvents, or from genetic engineering methods such as amplification, cloning and subcloning, the sequences of the invention being able to be carried by vectors.
• the nucleotide sequence SEQ ID No. 1 was obtained by sequencing the genome of Pyrococcus abyssi strain ORSAY by a method comprising a first step of sequencing of paired ends (random phase), followed by directed sequencing ensuring the completion of the sequence and assembling these sequences of nucleotide fragments using software (cf. Examples).
• sequence SEQ ID N ° 1 it is possible that it does not perfectly represent 100% the nucleotide sequence of the genome of Pyrococcus abyssi strain ORSAY and that some rare sequencing errors or indeterminacies remain in the sequence SEQ ID No. 1.
• nucleotide sequence homologous to a given sequence within the meaning of the present invention means a percentage of identity after optimal alignment with the bases of the given sequence of at least 80%, preferably 85%, 90%, 95% and 98%, this percentage being purely statistical and the differences between the two nucleotide sequences which can be distributed randomly and over their entire length.
• 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 randomly and over their entire length.
• 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], using the local homology algorithm of Neddleman and Wunsch (1970) [J. Mol. Biol. 48: 443], using the similarity search method of Pearson and Lipman (1988) [Proc. Natl. Acad. Sci.
• the percentage of identity between two nucleic acid or amino acid sequences is determined by comparing these two optimally aligned sequences per comparison window in which the region of the nucleic acid or amino acid sequence to be compared. may include additions or deletions with respect 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 in the comparison window. and multiplying the result obtained by 100 to obtain the percentage of identity between these two sequences.
• BLAST 2 sequences (Tatusova et al., "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol. Lett. 174: 247-250) available on the site http://www.ncbi.nlm.nih.gov/gorf/bl2.html.
• the parameters used being those given by default (in particular for the parameters “open gap penaltie”: 5, and “extension gap penaltie”: 2; the matrix chosen being for example the matrix “BLOSUM 62” proposed by the program), the percentage of identity between the two sequences to be compared being calculated directly through the program.
• Said homologous sequences exhibiting a percentage of identity with the bases of the nucleotide sequence SEQ ID No 1 of at least 80%, preferably 85%, 90%, 95% and 98% may for example include the sequences corresponding to the genomic sequence or the sequences of its fragments representative of bacteria belonging to the variants of the species Pyrococcus abyssi strain ORSAY.
• the terms family and genus are interchangeable with one another, the terms variant, strain and subspecies are also interchangeable with one another.
• These homologous sequences can thus correspond to variations linked to mutations within the same species or between species and correspond in particular to truncations, substitutions, deletions and / or additions of at least one nucleotide.
• Said homologous sequences can also correspond to variations linked to the degeneration of the genetic code or to a bias in the specific genetic code of the genus, of the species or of variant and which are likely to be present in Pyrococcus abyssi.
• nucleotide sequence complementary to a sequence of the invention is meant any DNA whose nucleotides are complementary to those of the sequence of the invention, and whose orientation is reversed (antiparallel or antisense sequence).
• fragments representative of the sequences according to the invention is intended to denote any nucleotide fragment having at least 15 nucleotides, preferably at least 30, 75, 150, 300, and 450 consecutive nucleotides of the sequence from which it is derived.
• Hybridization under stringent conditions 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 nucleotide fragments described above are advantageously as follows.
• the hybridization is carried out at a preferred temperature of 65 ° C. in the presence of SSC buffer, 1 ⁇ SSC corresponding to 0.15 M NaCl and 0.05 M Na citrate.
• the washing steps can, for example, be as follows:
• the intermediate stringency conditions using for example a temperature of 60 ° C. in the presence of a 5 ⁇ SSC buffer, or of low stringency, for example a temperature of 50 ° C in the presence of a 5 x SSC buffer, require respectively for the hybridization between the two sequences a less important overall complementarity.
• ORF sequences ORF (“open reading frame”)
• ORF open reading frame
• polypeptides preferably at least 100 amino acids, such as for example, but not limited to, the ORF sequences which will be described below.
• the numbering of the ORF nucleotide sequences which will subsequently be used in the present description corresponds to the numbering of the amino acid sequences of the proteins encoded by said ORFs.
• the nucleotide sequences ORF 2, ORF 3, ..., ORF 811 and ORF 812 respectively code for the proteins of amino acid sequences SEQ ID N ° 2, SEQ ID N ° 3, ..., SEQ ID N ° 811 and SEQ ID N ° 812 appearing in the list of sequences below.
• the detailed nucleotide sequences of the sequences ORF 2, ORF 3, ..., ORF 790 and ORF 791 are determined by their respective positions on the genomic sequence SEQ ID N ° 1 appearing in the list of sequences as well as by the characteristic "complement” indicating, if it is mentioned, that these sequences are located on the complementary strand of the sequence SEQ ID No. 1.
• 1765176 - nt 1764523 and the characteristic ⁇ complement> mentioned in section ⁇ 222> associated with the section " ⁇ 223> SEQ ID: 374" is the complementary sequence to the sequence between the positions nt 1765176 - nt 1764523, ends included , of the sequence SEQ ID No. 1; the detailed nucleotide sequence of ORF 2, identified by its positions nt 25162 - nt 26181 mentioned in section ⁇ 222> associated with the section " ⁇ 223> SEQ ID: 2", is the sequence between positions nt 25162 - nt 26181, ends included, of the sequence SEQ ID No. 1.
• 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, these methods are in particular described in the book by Sambrook et al., 1989. These representative fragments can also be obtained by chemical synthesis when their size is not too large and according to methods well known to those skilled in the art.
• the representative fragments according to the invention may be used, for example as a primer, to reconstitute some of said representative fragments, in particular those of which a part of the sequence is likely to be missing or imperfect, by methods well known in the art. skilled in the art such as amplification, cloning or sequencing techniques.
• 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 for the expression of polypeptide, in particular leading to a modification of the level of expression or of the activity of said polypeptide.
• modified nucleotide sequence will also be understood to mean any nucleotide sequence coding for a modified polypeptide as defined below.
• the subject of the present invention is nucleotide sequences of Pyrococcus abyssi characterized in that they are chosen from sequences ORF 2 to ORF 812. The numbering of the ORF sequences corresponds to the order of appearance of these sequences on the chromosome, the exception of ORF 792 to ORF 812.
• sequences ORF 2 to ORF 791 are defined as indicated above.
• the detailed nucleotide nucleotide sequences of the sequences ORF 792 to ORF 800 are defined by the position of the first nucleotide and of the last nucleotide on the genomic sequence SEQ ID No. 1 mentioned in the digital identifier ⁇ 223> of the corresponding peptide sequence SEQ ID.
• the detailed nucleotide sequences of the sequences ORF 792 to ORF 800 are defined by the position of the first nucleotide and of the last nucleotide on the genomic sequence SEQ ID No. 1 mentioned in the digital identifier ⁇ 223> of the corresponding peptide sequence SEQ ID.
• ORF 801, ORF 805, ORF 806, ORF 807, ORF 808, ORF 811 and ORF 812 are defined in Examples 3 to 7 below by the position of the first nucleotide and of the last nucleotide on the sequence genomics SEQ ID N ° 1.
• ORF 802, ORF 803, ORF 804, ORF 809 and ORF 810 can be identified in Examples 3 and 7 by deduction, from ORF 801 and ORF 808 from which they originate.
• the invention also relates to the nucleotide sequences characterized in that they comprise a nucleotide sequence chosen from: a) a nucleotide sequence ORF 2 to ORF 812 according to the invention; b) a homologous nucleotide sequence comprising at least 80% identity with a nucleotide sequence ORF 2 to ORF 812 according to the invention or as defined in a); c) a complementary nucleotide or RNA sequence corresponding to an ORF 2 to ORF 812 sequence according to the invention or as defined in a) or b); d) a nucleotide sequence of a fragment representative of an ORF 2 to ORF 812 sequence according to the invention or of a sequence as defined in a), b) or c); e) a nucleotide sequence capable of being obtained from an ORF 2 to ORF 812 sequence according to the invention or as defined in a), b), c) or d); and f) a nucleotide
• the homologous sequences are preferred having a percentage of identity with the bases of one of the nucleotide sequences ORF 2 to ORF 812 of at least 80%, preferably 90%, 95% and 98%.
• Said homologous sequences can comprise, for example, the sequences corresponding to the ORF sequences of bacteria belonging to the variants of the species Pyrococcus abyssi. These homologous sequences can correspond to variations linked to mutations within the same species or between species and correspond in particular to truncations, substitutions, deletions and / or additions of at least one nucleotide. Said homologous sequences can also correspond to variations linked to the degeneration of the genetic code or to a bias in the specific genetic code of the genus, of the species or of variant and which are likely to be present in Pyrococcus.
• the invention comprises the polypeptides encoded by a nucleotide sequence according to the invention, preferably by a fragment representative of the sequence SEQ ID No. 1 and corresponding to an ORF sequence, in particular the polypeptides of Pyrococcus abyssi, characterized in that they are chosen from the sequences SEQ ID No. 2 to SEQ ID No. 812.
• the invention also comprises the polypeptides characterized in that they comprise a polypeptide chosen from: a) a polypeptide according to the invention; b) a polypeptide homologous to a polypeptide according to the invention, or as defined in a); c) a fragment of at least 5 amino acids of a polypeptide according to the invention, or as defined in a) or b); d) a biologically active fragment of a polypeptide according to the invention, or as defined in a), b) or c); and e) a modified polypeptide of a polypeptide according to the invention, or as defined in a), b), c) or d).
• the terms polypeptide, peptide and protein are interchangeable.
• 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 else by chemical synthesis and that they can then comprise non-natural amino acids, as will be described below.
• homologous polypeptide is intended to denote the polypeptides having, with respect to the natural polypeptide, certain modifications such as in particular a deletion, addition or substitution of at least one amino acid, a truncation, an elongation, a chimeric fusion, and / or a mutation, or polypeptides with post-translational modifications.
• homologous polypeptides those whose amino acid sequence has at least 80%, preferably 90%, 95% and 98%, of homology with the amino acid sequences of the polypeptides according to the invention are preferred.
• substitution one or more consecutive or non-consecutive amino acids are replaced by “equivalent” amino acids.
• equivalent amino acid is intended here to denote 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 defined by the following. These equivalent amino acids can be determined either on the basis of their structural homology with the amino acids for which they are substituted, or on results of comparative tests of biological activity between the various polypeptides capable of being carried out.
• substitutions which may be carried out without resulting in a profound modification of the biological activity of the corresponding modified polypeptides, the replacements, for example, of leucine by valine or l isoleucine, aspartic acid by glutamic acid, glutamine by asparagine, arginine by lysine etc., the reverse substitutions being naturally possible under the same conditions.
• homologous polypeptides also correspond to the polypeptides encoded by the homologous nucleotide sequences as defined above and thus include in this definition the polypeptides mutated or corresponding to inter- or intra-species variations, which may exist in Pyrococcus, and which correspond in particular to truncations, substitutions, deletions and / or additions of at least one amino acid residue.
• biologically active fragment of a polypeptide according to the invention is intended to denote in particular a fragment of polypeptide, as defined below, having at least one of the characteristics of the polypeptides according to the invention, in particular in that it is capable of generally carrying out an activity, even partial, such as for example: - an enzymatic (metabolic) activity or an activity which may be involved in the biosynthesis or biodegradation of organic or inorganic compounds;
• an activity of transport (of energy, of ion), in the secretion; or - an activity in the process of replication, amplification, repair, transcription, translation or maturation, in particular of DNA or RNA.
• polypeptide fragment according to the invention is intended to denote a polypeptide comprising at least 5 amino acids, preferably 10, 15, 25, 50, 100 and 150 amino acids.
• the polypeptide fragments according to the invention may correspond to fragments isolated or purified naturally present in Pyrococcus abyssi or secreted by Pyrococcus abyssi, or correspond to fragments which can be obtained by cleavage of said polypeptide by a proteolytic enzyme, such as trypsin or chymotrypsin or collagenase, or by a chemical reagent, such as cyanogen bromide (CNBr) or by placing said polypeptide in a very acidic environment, for example at pH 2.5.
• a proteolytic enzyme such as trypsin or chymotrypsin or collagenase
• a chemical reagent such as cyanogen bromide (CNBr) or by placing said polypeptide in a very acidic environment, for example at pH 2.5.
• polypeptide fragments can also be prepared indifferently by chemical synthesis, from hosts transformed by an expression vector according to the invention containing a nucleic acid allowing the expression of said fragments, placed under the control of regulatory elements and / or appropriate expression.
• modified polypeptide of a polypeptide according to the invention is meant a polypeptide obtained by genetic recombination or by chemical synthesis as will be described below, having at least one modification with respect to the normal sequence. These modifications may in particular relate to amino acids at the origin of a specificity or of 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 'invention.
• polypeptides of equivalent, increased or decreased activity, and of equivalent specificity, narrower or wider are examples of the modified polypeptides.
• the modifications of the polypeptide will in particular aim:
• Chemical synthesis also has the advantage of being able to use non-natural amino acids, or non-peptide bonds.
• non-natural amino acids for example in D form, or else amino acid analogs, in particular sulfur-containing forms. example.
• nucleotide sequences encoding a polypeptide according to the invention also form part of the invention.
• the invention also relates to the nucleotide sequences according to the invention, characterized in that they code for a polypeptide of Pyrococcus abyssi, or one of its fragments, involved in the production or conversion of energy and in that they comprise a nucleotide sequence chosen from the following sequences: ORF 109, ORF 110, ORF 111, ORF 112, ORF 113, ORF 12, ORF 124, ORF 13, ORF 138, ORF 140, ORF 163, ORF 164, ORF 165, ORF 166, ORF 167, ORF 168, ORF 169, ORF 179, ORF 192, ORF 193, ORF 200, ORF 215, ORF 216, ORF 217, ORF 218, ORF 236, ORF 277, ORF 295, ORF 296 , ORF 297, ORF 32, ORF 364, ORF 369, ORF 385, ORF 386, ORF 387, ORF 400, ORF 40
• the subject of the invention is nucleotide sequences, characterized in that they code for a polypeptide of Pyrococcus abyssi, or one of its fragments, involved in the metabolism or transport of amino acids, and in that they comprise a nucleotide sequence chosen from the following sequences: ORF 115, ORF 126, ORF 128, ORF 135, ORF 172, ORF 178, ORF 180, ORF 183, ORF 184, ORF 187, ORF 196, ORF 199, ORF 20, ORF 202, ORF 203, ORF 205, ORF 210, ORF 211, ORF 212, ORF 24, ORF 243, ORF 247, ORF 25, ORF 26, ORF 27, ORF 284, ORF 291, ORF 30, ORF 304, ORF 305, ORF 307, ORF 308, ORF 309, ORF 31, ORF 310, ORF 321, ORF 377, ORF 378, ORF 38, ORF 3
• the invention also relates to the nucleotide sequences according to the invention, characterized in that they code for a polypeptide of Pyrococcus abyssi, or one of its fragments, involved in the metabolism or transport of the nucleotides, and in what they include a nucleotide sequence chosen from the sequences:
• the invention also relates to the nucleotide sequences according to the invention, characterized in that they code for a polypeptide of Pyrococcus abyssi, or one of its fragments, involved in the metabolism or the transport of carbohydrates, and in that they comprise a nucleotide sequence chosen from the following sequences: ORF 100, ORF 127, ORF 162, ORF 174, ORF 328, ORF 367, ORF 39, ORF 609, ORF 762, and one of their representative fragments.
• the invention also relates to the nucleotide sequences according to the invention, characterized in that they code for a pyrococcus abyssi polypeptide, or one of its fragments, involved in the metabolism of coenzymes, and in that they comprise a nucleotide sequence chosen from the following sequences: ORF 191, ORF 3, ORF 300, ORF 33, ORF 341, ORF 370, ORF 411, ORF 430, ORF 453, ORF 473, ORF 480, ORF 499, ORF 51, ORF 525, ORF 526, ORF 542 , ORF 602, ORF 608, ORF 615, ORF 637, ORF 641, ORF 653, ORF 667, ORF 715, ORF 725, ORF 732, ORF 740, ORF 746, ORF 749.0RF 750, ORF 768, ORF 785, and a of their representative fragments.
• the invention also relates to the nucleotide sequences according to the invention, characterized in that they code for a polypeptide of Pyrococcus abyssi, or one of its fragments, involved in the metabolism of lipids, and in that they comprise a nucleotide sequence chosen from the following sequences: ORF 312, ORF 313, ORF 545, ORF 558, ORF 559, and one of their representative fragments.
• the invention also relates to the nucleotide sequences according to the invention, characterized in that they code for a polypeptide of Pyrococcus abyssi, or one of its fragments, involved in the translation or in the structure or biogenesis of ribosomes, and in that they comprise a nucleotide sequence chosen from the following sequences:
• ORF 104 ORF 117, ORF 119, ORF 120, ORF 121, ORF 122, ORF 123, ORF 125, ORF 131, ORF 142, ORF 144, ORF 149, ORF 15, ORF 150, ORF 160, ORF 161, ORF 177 , ORF 190, ORF 206, ORF 221, ORF 245, ORF 253, ORF 285, ORF 290, ORF 302, ORF 318, ORF 351, ORF 358, ORF 374, ORF 375, ORF 379, ORF 384, ORF 389, ORF 412, ORF 416, ORF 428, ORF 431, ORF 476, ORF 479, ORF 496, ORF 541, ORF 547, ORF 549, ORF 561, ORF 579, ORF 6, ORF 605, ORF 623, ORF 649, ORF 675, ORF 676, ORF 677, ORF 678, ORF 679, ORF
• the invention also relates to the nucleotide sequences according to the invention, characterized in that they code for a polypeptide of Pyrococcus abyssi, or one of its fragments, involved in transcription, and in that they comprise a nucleotide sequence chosen from the following sequences: ORF 148, ORF 357, ORF 483, ORF 497, ORF 523, ORF 524, ORF 620, ORF 752, and one of their representative fragments.
• the invention also relates to the nucleotide sequences according to the invention, characterized in that they code for a polypeptide of Pyrococcus abyssi, or one of its fragments, involved in the replication, repair and recombination, and in that they comprise a nucleotide sequence chosen from the following sequences:
• ORF 139 ORF 158, ORF 197, ORF 198, ORF 209, ORF 219, ORF 246, ORF 280, ORF
• ORF 7 ORF 700, ORF 717, ORF 73, ORF 736, ORF 738, ORF 766, ORF 774, ORF 780,
• ORF 99 ORF 801, ORF 812, and one of their representative fragments.
• the invention also relates to the nucleotide sequences according to the invention, characterized in that they code for a polypeptide of Pyrococcus abyssi, or one of its fragments, involved in the synthesis of the outer membrane or of the wall cell, and in that they comprise a nucleotide sequence chosen from the following sequences:
• ORF 11 ORF 129, ORF 130, ORF 220, ORF 260, ORF 261, ORF 262, ORF 264, ORF 266, ORF 270, ORF 272, ORF 274, ORF 293, ORF 327, ORF 338, ORF 342, ORF 360 ,
• the invention also relates to the nucleotide sequences according to the invention, characterized in that they code for a pyrococcus abyssi polypeptide or one of its fragments involved in secretion or motility, and in that they comprise a nucleotide sequence chosen from the following sequences:
• ORF 102 ORF 189, ORF 214, ORF 275, ORF 326, ORF 332, ORF 352, ORF 381, ORF
• the invention also relates to the nucleotide sequences according to the invention, characterized in that they code for a chaperone protein of
• Pyrococcus abyssi or a fragment thereof, and in that they comprise a nucleotide sequence chosen from the following sequences:
• the invention also relates to the nucleotide sequences according to the invention, characterized in that they code for a polypeptide of Pyrococcus abyssi, or one of its fragments, involved in the metabolism and the transport of ions inorganic, and in that they comprise a nucleotide sequence chosen from the following sequences:
• ORF 105 ORF 114, ORF 134, ORF 19, ORF 208, ORF 226, ORF 227, ORF 229, ORF
• the invention also relates to the nucleotide sequences according to the invention, characterized in that they code for a polypeptide of Pyrococcus abyssi, or one of its fragments, with polymerase, DNA or RNA polymerase activity, and in that that they comprise a nucleotide sequence chosen from the following sequences:
• the invention also relates to the nucleotide sequences according to the invention, characterized in that they code for a polypeptide of Pyrococcus abyssi, or one of its fragments, with transferase activity, and in that they comprise a nucleotide sequence chosen from the following sequences:
• the invention also relates to the nucleotide sequences according to the invention, characterized in that they code for a polypeptide of Pyrococcus abyssi, or one of its fragments, with hydrolase activity, and in that they comprise a nucleotide sequence chosen from the following sequences: ORF 10, ORF 116, ORF 233, ORF 240, ORF 337, ORF 350, ORF 353, ORF 440, ORF
• the invention also relates to the nucleotide sequences according to the invention, characterized in that they encode a polypeptide of Pyrococcus abyssi, or one of its fragments, comprising an intern, and in that they comprise a nucleotide sequence chosen from the following sequences:
• ORF 132 ORF 16, ORF 250, ORF 340, ORF 484, ORF 603, ORF 761, ORF 801, ORF
• the invention also relates to the nucleotide sequences according to the invention, characterized in that they code for a polypeptide of Pyrococcus abyssi, or one of its fragments, with isomerase activity, and in that they comprise a nucleotide sequence chosen from the following sequences: ORF 176, ORF 282, ORF 283, ORF 399, ORF 426, ORF 531, ORF 583, ORF 688, and their representative fragments.
• the invention also relates to the nucleotide sequences according to the invention, characterized in that they code for a polypeptide of Pyrococcus abyssi, or one of its fragments, with ligase activity, and in that they comprise a nucleotide sequence chosen from the following sequences:
• ORF 28 ORF 639, ORF 65, and their representative fragments.
• the invention also relates to the nucleotide sequences according to the invention, characterized in that they code for a polypeptide of Pyrococcus abyssi, or one of its fragments, with lyase activity, and in that they comprise a nucleotide sequence chosen from the following sequences:
• ORF 156 ORF 223, ORF 271, ORF 306, ORF 36, ORF 362, ORF 403, ORF 472, ORF
• the invention also relates to the nucleotide sequences according to the invention, characterized in that they code for a polypeptide of Pyrococcus abyssi, or one of its fragments, with phosphatase activity, and in that they comprise a nucleotide sequence chosen from the following sequences:
• ORF 18 ORF 354, ORF 398, ORF 75, ORF 760, ORF 793, and their representative fragments.
• the invention also relates to the nucleotide sequences according to the invention, characterized in that they code for a polypeptide of
• Pyrococcus abyssi or a fragment thereof, involved in the metabolism of sugars, and in that they comprise a nucleotide sequence chosen from the following sequences:
• the invention also relates to the nucleotide sequences according to the invention, characterized in that they code for a polypeptide of
• Pyrococcus abyssi or a fragment thereof, with protease activity, and in that they comprise a nucleotide sequence chosen from the following sequences: ORF 143, ORF 21, ORF 256, ORF 433, ORF 437, ORF 471, ORF 521, ORF 560, ORF 584, ORF 626, ORF 699, ORF 705, ORF 9, ORF 97, ORF 794, and their representative fragments.
• nucleotide sequences according to the invention characterized in that they code for a pyrococcus abyssi polypeptide or one of its fragments with activity:
• the subject of the invention is polypeptides according to the invention, characterized in that they comprise a polypeptide involved in the production or the conversion of energy chosen from the polypeptides of following sequences: SEQ ID N ° 109, SEQ ID N ° 110, SEQ ID N ° 111, SEQ ID N ° 112, SEQ ID N ° 113, SEQ ID N ° 12, SEQ ID N ° 124, SEQ ID N ° 13, SEQ ID N ° 138, SEQ ID N ° 140, SEQ ID N ° 163, SEQ ID N ° 164, SEQ ID N ° 165, SEQ ID N ° 166, SEQ ID N ° 167, SEQ ID N ° 168, SEQ ID N ° 169 , SEQ ID N ° 179, SEQ ID N ° 192, SEQ ID N ° 193, SEQ ID N ° 200, SEQ ID N ° 215, SEQ ID N ° 216, SEQ ID N
• the invention also relates to polypeptides according to the invention, characterized in that they comprise a polypeptide involved in the metabolism or transport of amino acids chosen from the polypeptides of the following sequences: SEQ ID N ° 115 , SEQ ID N ° 126, SEQ ID N ° 128, SEQ ID N ° 135, SEQ ID N ° 172, SEQ ID N ° 178, SEQ ID N ° 180, SEQ ID N ° 183, SEQ ID N ° 184, SEQ ID N ° 187, SEQ ID N ° 196, SEQ ID N ° 199, SEQ ID N ° 20, SEQ ID N ° 202, SEQ ID N ° 203, SEQ ID N ° 205, SEQ ID N ° 210, SEQ ID N ° 211, SEQ ID N ° 212, SEQ ID N ° 24, SEQ ID N ° 243, SEQ ID N ° 247, SEQ ID N ° 25, SEQ ID N
• the invention also relates to polypeptides according to the invention, characterized in that they comprise a polypeptide involved in the metabolism or the transport of nucleotides chosen from the polypeptides of the following sequences: SEQ ID No. 101, SEQ ID N ° 14, SEQ ID N ° 155, SEQ ID N ° 181, SEQ ID N ° 182, SEQ ID N ° 185, SEQ ID N ° 22, SEQ ID N ° 224, SEQ ID N ° 23, SEQ ID N ° 244, SEQ ID N ° 251, SEQ ID N ° 257, SEQ ID N ° 289, SEQ ID N ° 335, SEQ ID N ° 344, SEQ ID N ° 345, SEQ ID N ° 359, SEQ ID N ° 368, SEQ ID N ° 373, SEQ ID N ° 394, SEQ ID N ° 395, SEQ ID N ° 415, SEQ ID N ° 417, SEQ ID N
• the invention also relates to polypeptides according to the invention, characterized in that they comprise a polypeptide involved in the metabolism or the transport of carbohydrates chosen from the polypeptides of the following sequences:
• the invention also relates to polypeptides according to the invention, characterized in that they comprise a polypeptide involved in the metabolism of coenzymes chosen from the polypeptides of the following sequences: SEQ ID No.
• SEQ ID N ° 3 SEQ ID N ° 300, SEQ ID N ° 33, SEQ ID N ° 341, SEQ ID N ° 370, SEQ ID N ° 411, SEQ ID N ° 430, SEQ ID N ° 453, SEQ ID N ° 473 , SEQ ID N ° 480, SEQ ID N ° 499, SEQ ID N ° 51, SEQ ID N ° 525, SEQ ID N ° 526, SEQ ID N ° 542, SEQ ID N ° 582, SEQ ID N ° 602, SEQ ID N ° 608, SEQ ID N ° 615, SEQ ID N ° 637, SEQ ID N ° 641, SEQ ID N ° 653, SEQ ID N ° 667, SEQ ID N ° 715, SEQ ID N ° 725, SEQ ID N ° 732, SEQ ID N ° 740, SEQ ID N ° 746, SEQ ID N ° 749, SEQ ID NO
• the invention also relates to polypeptides according to the invention, characterized in that they comprise a polypeptide involved in lipid metabolism chosen from the polypeptides of the following sequences: SEQ ID No. 312, SEQ ID N ° 313, SEQ ID N ° 545, SEQ ID N ° 558, SEQ ID N ° 559, and one of their fragments.
• the invention also relates to polypeptides according to the invention, characterized in that they comprise a polypeptide involved in the translation or in the structure or biogenesis of the ribosomes chosen from the polypeptides of the following sequences: SEQ ID N ° 104, SEQ ID N ° 117, SEQ ID N ° 119, SEQ ID N ° 120, SEQ ID N ° 121, SEQ ID N ° 122, SEQ ID N ° 123, SEQ ID N ° 125, SEQ ID N ° 131, SEQ ID N ° 142, SEQ ID N ° 144, SEQ ID N ° 149, SEQ ID N ° 15, SEQ ID N ° 150, SEQ ID N ° 160, SEQ ID N ° 161, SEQ ID N ° 177, SEQ ID N ° 190, SEQ ID N ° 206, SEQ ID N ° 221, SEQ ID N ° 245, SEQ ID N ° 253, SEQ ID N ° 186
• the invention also relates to polypeptides according to the invention, characterized in that they comprise a polypeptide involved in the transcription chosen from the polypeptides of the following sequences: SEQ ID No. 148, SEQ ID No. 357 , SEQ ID N ° 483, SEQ ID N ° 497, SEQ ID N ° 523, SEQ ID N ° 524, SEQ ID N ° 620, SEQ ID N ° 752, and one of their fragments.
• the invention also relates to polypeptides according to the invention, characterized in that they comprise a polypeptide involved in replication, repair and recombination, chosen from the polypeptides of the following sequences: SEQ ID N ° 139, SEQ ID N ° 158, SEQ ID N ° 197, SEQ ID N ° 198, SEQ ID N ° 209, SEQ ID N ° 219, SEQ ID N ° 246, SEQ ID N ° 280, SEQ ID N ° 281, SEQ ID N ° 292, SEQ ID N ° 294, SEQ ID N ° 316, SEQ ID N ° 323, SEQ ID N ° 34, SEQ ID N ° 347, SEQ ID N ° 35, SEQ ID N ° 376, SEQ ID N ° 419, SEQ ID N ° 42, SEQ ID N ° 421, SEQ ID N ° 425, SEQ ID N ° 455, SEQ ID N ° 468, SEQ ID
• the invention also relates to polypeptides according to the invention, characterized in that they comprise a polypeptide involved in the synthesis of the outer membrane or of the cell wall, chosen from the polypeptides of the following sequences:
• the invention also relates to polypeptides according to the invention, characterized in that they comprise a polypeptide involved in secretion or motility, chosen from the polypeptides of the following sequences:
• the invention also relates to polypeptides according to the invention, characterized in that they comprise a chaperone protein chosen from the proteins of the following sequences: * SEQ ID No. 170, SEQ ID No. 171, SEQ ID N ° 252, SEQ ID N ° 372, SEQ ID N ° 454, SEQ
• the invention also relates to polypeptides according to the invention, characterized in that they comprise a polypeptide involved in the metabolism and the transport of inorganic ions, chosen from the polypeptides of the following sequences:
• the invention also relates to polypeptides according to the invention, characterized in that they comprise a polypeptide with polymerase activity, DNA or RNA polymerase, chosen from the polypeptides of the following sequences: SEQ ID No. 145, SEQ ID N ° 146, SEQ ID N ° 147, SEQ ID N ° 355, SEQ ID N ° 363, SEQ ID N ° 485, SEQ ID N ° 720, SEQ ID N ° 721, SEQ ID N ° 722, SEQ ID N ° 723, SEQ ID N ° 737, SEQ ID N ° 776, SEQ ID N ° 807, SEQ ID N ° 808, SEQ ID N ° 809, SEQ ID N ° 812, and their fragments.
• polypeptides chosen from the polypeptides of the following sequences: SEQ ID No. 145, SEQ ID N ° 146, SEQ ID N ° 147, SEQ ID N ° 355, SEQ ID N ° 36
• the invention also relates to polypeptides according to the invention, characterized in that they comprise a polypeptide with transferase activity chosen from the polypeptides of the following sequences:
• the invention also relates to polypeptides according to the invention, characterized in that they comprise a polypeptide with hydrolase activity chosen from the polypeptides of the following sequences:
• SEQ ID N ° 10 SEQ ID N ° 10
• SEQ ID N ° 116 SEQ ID N ° 233
• SEQ ID N ° 240 SEQ ID N ° 337
• SEQ ID N ° 350 SEQ ID N ° 353, SEQ ID N ° 440, SEQ ID N ° 593, SEQ ID N ° 691, SEQ ID N ° 692, SEQ ID N ° 693, SEQ ID N ° 697, SEQ ID N ° 764, SEQ ID N ° 85, and their fragments.
• the invention also relates to polypeptides according to the invention, characterized in that they comprise a polypeptide comprising an intein chosen from the polypeptides of the following sequences: SEQ ID No. 132, SEQ ID No. 16, SEQ ID N ° 250, SEQ ID N ° 340, SEQ ID N ° 484, SEQ ID N ° 603, SEQ ID N ° 761, SEQ ID N ° 801, SEQ ID N ° 803, SEQ ID N ° 804, SEQ ID No. 808, SEQ ID No. 810, and their fragments.
• polypeptides comprising an intein chosen from the polypeptides of the following sequences: SEQ ID No. 132, SEQ ID No. 16, SEQ ID N ° 250, SEQ ID N ° 340, SEQ ID N ° 484, SEQ ID N ° 603, SEQ ID N ° 761, SEQ ID N ° 801, SEQ ID N ° 803, SEQ ID N ° 804, SEQ ID No
• the invention also relates to polypeptides according to the invention, characterized in that they comprise a polypeptide with isomerase activity chosen from the polypeptides of the following sequences: SEQ ID N ° 176, SEQ ID N ° 282, SEQ ID N ° 283, SEQ ID N ° 399, SEQ ID N ° 426, SEQ ID N ° 531, SEQ ID N ° 583, SEQ ID N ° 688, and their fragments.
• the invention also relates to polypeptides according to the invention, characterized in that they comprise a polypeptide with ligase activity chosen from the polypeptides of the following sequences:
• the invention also relates to polypeptides according to the invention, characterized in that they comprise a polypeptide with lyase activity chosen from the polypeptides of the following sequences: SEQ ID No. 156, SEQ ID No. 223, SEQ ID N ° 271, SEQ ID N ° 306, SEQ ID N ° 36, SEQ ID N ° 362, SEQ ID N ° 403, SEQ ID N ° 472, SEQ ID N ° 517, SEQ ID N ° 650, SEQ ID N ° 796, and their fragments.
• polypeptides chosen from the polypeptides of the following sequences: SEQ ID No. 156, SEQ ID No. 223, SEQ ID N ° 271, SEQ ID N ° 306, SEQ ID N ° 36, SEQ ID N ° 362, SEQ ID N ° 403, SEQ ID N ° 472, SEQ ID N ° 517, SEQ ID N ° 650, SEQ ID N ° 796, and their fragments
• the invention also relates to polypeptides according to the invention, characterized in that they comprise a polypeptide with phosphatase activity chosen from the polypeptides of the following sequences:
• the invention also relates to polypeptides according to the invention, characterized in that they comprise a polypeptide involved in the metabolism of sugars chosen from the polypeptides of the following sequences:
• the invention also relates to polypeptides according to the invention, characterized in that they comprise a polypeptide with protease activity chosen from the polypeptides of the following sequences:
• polypeptides according to the invention characterized in that they comprise a polypeptide with activity:
• nucleotide sequences and the polypeptides according to the present invention the nucleotide sequences coding for the polypeptides and the polypeptides with polymerase or protease activity are preferred.
• the functional group to which a polypeptide of the invention belongs as well as its corresponding nucleotide sequence can be determined either by analogy compared with already known sequences, or by the use of standard biochemical techniques , cytology associated with genetic engineering techniques such as immunoaffinity, localization by immunostaining, differential extraction, measurement of enzymatic activity, study of the inducing or repressive activity of expression or the study of expression in E. coli.
• the nucleotide sequences ORF 2 to ORF 812
• amino acid sequences SEQ ID No. 2 to SEQ ID No. 812
• nucleotide sequence or an amino acid sequence mentioned in a given functional group, can also form part of another group taking into account for example of the inter-implication between listed groups, and of the base of the chosen sequences (such as the swissprot or COG library for the present invention).
• the present invention also relates to the nucleotide and / or polypeptide sequences according to the invention, characterized in that said sequences are recorded on a support, called a recording support, the shape and nature of which facilitate reading, analysis and exploitation of said sequences.
• a recording support the shape and nature of which facilitate reading, analysis and exploitation of said sequences.
• These supports may of course also contain other information extracted from the present invention such as in particular analogies with already known sequences, such as those mentioned in Table 1 of this description, and / or may also contain information concerning the nucleotide and / or polypeptide sequences of other microorganisms in order to facilitate comparative analysis and the exploitation of results obtained.
• recording media particular preference is given to media readable by a computer, such as magnetic, optical, electric or hybrid media such as, for example, floppy disks, CD-ROMs or recording cassettes. .
• the invention also relates to nucleotide sequences which can be used as primer or probe, characterized in that said sequences are chosen from the nucleotide sequences according to the invention.
• the invention further relates to the use of a nucleotide sequence according to the invention, as a primer or probe, for the amplification of nucleic acid sequences.
• nucleotide sequences according to the invention can thus be used to amplify nucleotide sequences, in particular by the PCR technique (polymerase chain reaction) (Erlich, 1989; Innis et al., 1990; Rolfs et al., 1991, and White et al., 1997).
• PCR technique polymerase chain reaction
• oligodeoxyribonucleotide or oligo-ribonucleotide primers advantageously have a length of at least 10 nucleotides, preferably at least 15 nucleotides, and even more preferably at least 20 nucleotides.
• nucleotide sequences of the invention in particular the primers according to the invention, can also be used in other methods of amplification of a target nucleic acid, such as:
• TAS Transcription-based Amplification System
• 3SR Self-Sustained Sequence Replication
• TMA Transcription Mediated Amplification
• polynucleotides of the invention can also be used in techniques for amplification or modification of the nucleic acid serving as a probe, such as: - the LCR (Ligase Chain Reaction) technique, described by Landegren et al. in 1988 and perfected by Barany et al. in 1991, which used a thermostable ligase;
• LCR Liigase Chain Reaction
• the invention also relates to the nucleotide sequences of fragments capable of being obtained by amplification using at least one primer according to the invention.
• the target polynucleotide is an RNA, for example a
• MRNA it is possible to use, prior to the implementation of an amplification reaction using at least one primer according to the invention or to the implementation of a detection method using at least one probe of the invention, an enzyme of reverse transcriptase type in order to obtain a cDNA from the RNA contained in the sample.
• the cDNA obtained will then serve as a target for the primer (s) or the probe (s) used in the amplification method according to the invention.
• the invention also relates to the nucleotide sequences of fragments capable of being obtained by hybridization using at least one probe according to the invention.
• the invention also includes the nucleotide sequences according to the invention, characterized in that they are immobilized on a support, covalently or non-covalently.
• Another object of the present invention is a vector for the cloning and / or expression of a sequence, characterized in that it contains a nucleotide sequence according to the invention.
• preferred are the vectors containing a nucleotide sequence coding for a pyrococcus abyssi polypeptide or one of its fragments.
• vectors containing a nucleotide sequence coding for a pyrococcus abyssi polypeptide, or a fragment thereof, involved in energy conversion or production, in metabolism and / or transport.
• amino acids amino acids, carbohydrates or sugars, lipids, nucleotides, inorganic ions or coenzymes, or involved in transcription, replication, repair or recombination, especially DNA or RNA , or in secretion or motility, biogenesis or the structure of ribosomes, the synthesis of cell wall or outer membrane, or coding for a polypeptide, or a fragment thereof, with isomerase, ligase, lyase, phosphatase, protease, transferase, polymerase, RNA or DNA polymerase activity, or for chaperone proteins or containing internins .
• vectors according to the invention preference is also given to vectors containing a nucleotide sequence coding for a pyrococcus abyssi polypeptide, or one of its fragments, of nuclear cell proliferation antigen type, of protein type related to a replication factor, of DNA binding protein type, transcription factor type, integrase / recombinase type or redox catalyst type.
• the vectors according to the invention characterized in that they comprise the elements allowing the expression and / or the secretion of said nucleotide sequences in a determined host cell, also form part of the invention.
• the vector must then include a promoter, translation initiation and termination signals, as well as suitable regions for transcription regulation. It must be able to be maintained stably in the host cell and may possibly have specific signals specifying the secretion of the translated protein. These different elements are chosen 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 integrative vectors of the chosen host.
• Such vectors will be prepared according to the methods commonly used by those skilled in the art, and the resulting clones may be introduced into an appropriate host by standard methods, such as for example lipofection, electroporation, thermal shock.
• the vectors according to the invention are for example vectors of plasmid or viral origin. These vectors are useful for transforming host cells in order to clone or express the nucleotide sequences of the invention.
• the invention also includes host cells transformed with a vector according to the invention.
• These cells can be obtained by introducing into host cells a nucleotide sequence inserted into a vector as defined above, then culturing said cells under conditions allowing replication and / or expression of the transfected nucleotide sequence.
• the cell host can be chosen from prokaryotic or eukaryotic systems, such as for example bacterial cells (Olins and Lee, 1993), but also cells yeast (Buckholz, 1993), as well as animal cells, in particular mammalian cell cultures (Edwards and Aruffo, 1993), and in particular Chinese hamster ovary (CHO) cells, but also d insects in which methods using baculoviruses can be used, for example (Luckow, 1993).
• a preferred host cell for the expression of the proteins of the invention consists of prokaryotic cells, such as Gram " bacteria.
• the invention also relates to animals, except humans, comprising one of said cells transformed according to the invention.
• transgenic animals according to the invention overexpressing one or more of the genes of Pyrococcus abyssi will preferably be carried out on rats, mice or rabbits according to methods well known to those skilled in the art such as by transfections, viral or non-viral.
• Transgenic animals overexpressing one or more of said genes may be obtained by transfection of multiple copies of said genes under the control of a powerful promoter of ubiquitous nature, or selective for a type of tissue.
• Transgenic animals can also be obtained by homologous recombination on embryonic stem cells, transfer of these stem cells to embryos, selection of the affected chimeras at the level of the reproductive lines, and growth of said chimeras.
• the transformed cells as well as the transgenic animals according to the invention can be used in processes for the preparation of recombinant polypeptide. It is now possible to produce recombinant polypeptides in relatively large quantities by genetic engineering using the cells transformed by expression vectors according to the invention or by using transgenic animals according to the invention.
• the methods for preparing a polypeptide of the invention in recombinant form characterized in that they use a vector and / or a cell transformed with a vector according to the invention and / or a transgenic animal comprising one of said cells transformed according to the invention, are themselves included in the present invention.
• the recombinant polypeptides obtained as indicated above can be both in glycosylated and non-glycosylated form and may or may not have the natural tertiary structure.
• a preferred variant consists in producing a recombinant polypeptide fused to a “carrier” protein (chimeric protein).
• carrier chimeric protein
• the invention relates to a process for the preparation of a polypeptide of the invention comprising the following steps: a) culture of the transformed cells under conditions allowing the expression of a recombinant polypeptide of nucleic acid sequence according to l invention; b) where appropriate, recovery of said recombinant polypeptide.
• the process for preparing a polypeptide of the invention implements a transgenic animal according to the invention, the recombinant polypeptide is then extracted from said animal.
• the subject of the invention is also a polypeptide capable of being obtained by a method of the invention as described above.
• the invention also includes a process for preparing a synthetic polypeptide, characterized in that it uses an amino acid sequence of polypeptides according to the invention.
• the invention also relates to a synthetic polypeptide obtained by a method according to the invention.
• polypeptides according to the invention can also be prepared by conventional techniques, in the field of peptide synthesis. This synthesis can be carried out in homogeneous solution or in solid phase. For example, we will use the synthesis technique in homogeneous solution described by Houbenweyl in 1974.
• the invention further relates to hybrid polypeptides having at least one polypeptide or one of its fragments according to the invention.
• the methods of synthesis of the hybrid molecules include the methods used in genetic engineering to construct hybrid nucleotide sequences coding for the polypeptide sequences sought.
• the invention also includes the vectors characterized in that they contain one of said hybrid nucleotide sequences.
• the host cells transformed by said vectors, the transgenic animals comprising one of said transformed cells as well as the methods for preparing recombinant polypeptides using said vectors, said transformed cells and / or said transgenic animals are of course also part of the invention.
• nucleotide sequences of the invention have been identified by homology with sequences known as coding for example for polypeptides or fragments of enzymatic polypeptides involved in the biosynthesis or biodegradation of organic or inorganic molecules . It is thus possible to use said polypeptides of the invention in a similar manner for the biosynthesis or the biodegradation of organic or inorganic compounds having an industrial or therapeutic interest.
• polypeptides mention may be made in particular of the enzymes involved in metabolism such as proteolytic enzymes, aminotransferases, of glucose metabolism, or the enzymes which can be used in the biosynthesis of sugars, amino acids, fatty acids, polypeptides, nucleotides, nucleic acids or any other organic or inorganic compound, or even enzymes which can be used in the biodegradation of organic or inorganic compounds.
• enzymes involved in metabolism such as proteolytic enzymes, aminotransferases, of glucose metabolism, or the enzymes which can be used in the biosynthesis of sugars, amino acids, fatty acids, polypeptides, nucleotides, nucleic acids or any other organic or inorganic compound, or even enzymes which can be used in the biodegradation of organic or inorganic compounds.
• mutated or modified enzymes corresponding to mutated or modified polypeptides according to the invention which may also be used for biosynthesis or biodegradation of organic or inorganic compounds on an industrial level, for example the production of compounds of interest, the reprocessing of processing residues applied to the agro-food industries, to the stationery or to the chemical, pharmaceutical or diagnostic industries.
• polypeptides with DNA or RNA polymerase activity according to the invention can advantageously be used in methods requiring the amplification of a nucleic acid, or in the uses, methods or applications mentioned in the examples.
• polypeptides with DNA or RNA polymerase activity according to the invention can be used in a process for the polymerization and / or amplification of target nucleic acid in a biological sample (tissue, cell or biological fluid), characterized in that that it comprises the following stage: a) bringing this biological sample into contact with a polypeptide, or one of its fragments, with DNA or RNA polymerase activity according to the invention under conditions allowing a polymerization reaction and / or d amplification between said polypeptide and acids nucleic acids present in the biological sample.
• the invention also relates to a reagent or a kit (or necessary) comprising a polypeptide, or a fragment thereof, with DNA or RNA polymerase activity according to the invention for the polymerization and / or amplification of target nucleic acid in a biological sample.
• Such reagents or kits may in particular be used for the detection and / or identification of target nucleic acid, in particular for the detection and / or identification of specific nucleic acid of pathogenic microorganism, such as bacteria , yeasts, parasites or viruses, or else for the detection and / or identification of genes of interest, or of mutations among these genes of interest, as well as for the uses, methods or applications mentioned in the examples below relating to DNA polymerase type I or II.
• Figure 1 Characterization of the two subunits of recombinant replication factor C obtained by co-expression in E.coli MC1061 (DE3)
• Figures 2A and 2B Characterization of the large subunit of factor C of recombinant replication by autoradiography of the immunoblot (Western Blot) with antibodies A ( Figure 2A) and B ( Figure 2B)
• RF-C A and RF-C B reaction with the large sub-unit of RF-C of calf thymus (control), line 1: fraction expressed in the insoluble part (pellet); lane 2: fraction expressed in the soluble fraction (supernatant); lane 3: soluble fraction of an uninduced clone.
• Figure 3 Characterization of the recombinant PCNA polypeptide
• Electrophoresis gel line 1: LMW; line 2: not induced; line 3: induced; line
• FIG. 4 represents the incorporation into 3H-TTP using the OligoF / lambda matrix as a function of the amount of polish with or without PCNA; 1: 2U of pol I; 2: 1U of pol I; 3:
• - line 1 LMW
• - lines 2, 3, 4 clones co-expressing the 2 subunits (polB and polC) of DNA polymerase II induced 15 h at 37 ° C after thermal denaturation 15 min at 80 ° C;
• Figure 6 Polymerase activity of DNA polymerase II as a function of the pH expressed as a percentage of the activity obtained for the optimal pH at 6.5.
• Figure 7 Characterization of the recombinant replication protein A expressed in E. coli B121 pLys S (DE3)
• Figure 8 Alignment of RPA sequences of Pyrococcus abyssi, Pyrococcus horikochii and RP70 of Saccharomyces cerevisiae and of human origin.
• Control E. coli BL21 (DE3) pLysS;
• the ORSAY Pyrococcus abyssi strain used is the strain deposited on April 9, 1999 at the National Collection of Culture of Microorganisms, INSTITUT PASTEUR, Paris, France, under the number 1-2180.
• the cells of Pyrococcus abyssi are harvested by centrifugation at low speed and at 4 ° C., according to the protocol described in the work by Sambrook et al., 1989.
• the DNA in soluble form is prepared according to the alkaline lysis method described in the work of Sambrook et al., 1989, followed by two phenol / chloroform extractions, two chloroform extractions, elimination of the RNA by treatment with RNAse then a concentration of DNA by ethanolic precipitation.
• the DNA is taken up in TRIS-EDTA (20 mM - 1 mM) and assayed.
• the DNA immobilized in an agarose block is prepared in the following manner: after a culture under the conditions described above up to an OD 600 nm of 0.3, the cells are cooled in ice and then recovered by centrifugation at low speed, at 4 ° C. The cell pellet is washed twice in the culture medium, once in 50 mM EDTA pH 8.0, once in 125 mM EDTA pH 8.0, and centrifuged after each washing. The cells are then resuspended in 125 mM EDTA pH 8.0 at 37 ° C and then an equal volume of low melting agarose (Sigma Type VII) prepared at 1.6% in deionized water and tempered at 37 ° C.
• the mixture is poured into 0.1 ml molds and left at room temperature to allow gelation of cell-agarose inclusions.
• the inclusion blocks are removed from the mold and treated in ESP buffer (0.5 M EDTA pH 9.0, 1% Lauroyl sarcosine and 1 mg / ml Proteinase K) for 48 hours, at 37 ° C, then stored at 4 ° C .
• the inclusion blocks are washed in TRIS-EDTA (10 mM - 1 mM) + 0.250 mg / ml PMSF, extensively (4 to 6 times 2 to 4 hours) before use.
• EXAMPLE 1 SEQUENCING STRATEGY
• the strategy adopted involves a random phase characterized by the sequencing of paired ends (Roach et al., Genomics, 26: 345, 1995), coming from clones selected at random, followed by a directed phase ensuring the completion of the sequence .
• the operation was carried out in 3 stages:
• a 20 ⁇ g sample of DNA was subjected to a partial digestion with the enzyme CviJI (CHIMERx-Ozyme), in a reaction volume of 300 ⁇ l.
• the incubation temperature was reduced to 15 ° C, to better control the progress of the reaction.
• Six 50 ⁇ l samples were taken (after 2 min, 5 min, 10 min, 20 min, 45 min and 90 min of incubation) and the reaction was immediately stopped by adding EDTA and SDS (20 mM and 0.2% final, respectively). After analysis by electrophoresis of an aliquot (2 ⁇ l), the samples corresponding to the states of partial digestion deemed adequate were pooled, their DNA precipitated with ethanol, then subjected to a preparative separation on low point agarose gel.
• CviJI CHIMERx-Ozyme
• a cosmid vector such as for example SuperCosl (Stratagene).
• 1635 end sequences were produced from these cosmid clones and allowed the extension of the assembly skeleton over longer distances. About 500 of them were performed on cosmids selected by hybridization to PCR probes from the ends of the contigs during assembly.
• the assembly was carried out using the PHRED and PHRAP software developed by P. Green et al. (Ewing et al., Génome Res., 8: 175, 1998) and the generated data exploited interactively using CONSED (Gordon et al., Génome Res., 8: 195, 1998).
• 14 cloning discontinuities remained, for about 98% sequential coverage.
• the comparison of the sequence data with the digestion profile of the genomic DNA for the enzyme NotI made it possible to extend the assembly skeleton over the entire molecule.
• the covering of these 14 cloning discontinuities (from 0.6 to 5 kb) as well as the strengthening of the weak bonds were carried out by sequencing of the products obtained by LA-PCR (Long and Acurate Polymerase Chain Reaction). 3. Finishing step
• the nucleotide primers were determined semi-automatically using the PRIMO software (Ping LI et al., Genomics, 40, 476-485, 1997).
• Direct sequencing on the P. abyssi genome has been used to resolve certain cloning gaps and confirm a few variable sites and suspected polymorphisms. Additional validation of the assembly carried out by comparison of the restriction map deduced from the assembled sequence with the digestion profile observed on the DNA of P. abyssi for the enzymes NotI and Ascl. A finer validation was carried out by “Southern blotting” of the DNA of the ORSAY strain digested by the enzymes BamHI, BgHI, EcoRI, HindIII, PstI eet Xbal, and hybrid with 33 probes obtained by LA6PCR, corresponding in particular to the 14 discontinuities of cloning observed.
• Example 2 ANNOTATION OF THE SEQUENCES
• the annotation of the sequence of Pyrococcus abyssi follows a simple scheme: determination of the coding segments, determination of coding regions by similarity, estimation of a statistical model of a coding region, evaluation of the coding potential of the different region, determination of the function of these segments by comparison with the sequences contained in the public domain sequence databases.
• a simple heuristic was used: it consists in determining all the maximum segments going from an initiating codon to a stop codon with a length greater than 100 sense codons. There are 2342 such open reading frames, which were then compared to the Swissprot protein sequence bank (Bairoch A and Apweiler R., Nucleic Acids Res.
• ftp // ftp .tigr .org / pub / data h influenzae / GHI .pep. gz. ftp: // ftp. tigr. org / pub / data / h_py lori / GHP .pep. gz, ftp: // ftp. tigr. org / pub / data / m J anashii / GMH .pep. gz, ftp: // ftp. tigr .org / pub / data / m_genitalium / GMG. pep. gz), and in cyanobase
• BLAST software public domain, Altschul et al., 1990 was used to search for homologies between a sequence and protein or nucleic databases. The main public databases were used.
• Table 1 List of putative functions of the proteins of sequences SEQ ID No. 1 to SEQ ID No. 791 encoded by their respective nucleotide sequence ORF 1 to ORF 758 of the genome of Pyrococcus abyssi ORSAY Legend of table 1:
• SEQ ID N ° 262 Glycosyltransferases, typically involved in cell wall biogenesis
• SEQ ID N ° 263 Carbonic anhydrases homologous to acetyltransferases of the isoleucine patch superfamily
• SEQ ID N ° 264 Predicted pyridoxal phosphate-dependent enzyme apparently involved in regulation of cell wall biogenesis
• SEQ ID N ° 265 Predicted dehydrogenases and related proteins
• NCAIR Phosphoribosylcarboxyaminoimidazole
• Threonine dehydratase / Threonine synthase SEQ ID No. 587 Glutamate 5-kinase
• SEQ ID No. 619 Succinyl-CoA synthetase, alpha subunit-related enzymes
• SEQ ID No. 627 Predicted permease SEQ ID No. 628 NTP pyrophosphohydrolases (MutT family) including oxidative damage repair enzymes
• SEQ ID N ° 668 ATPases involved in chromosome partitioning SEQ ID N ° 669 AAA superfamily and related ATPases
• ALKYLGLYCEROPHOSPHOCHOLINE ESTERASE (EC 3.1.1.) SEQ ID N ° 698 Dehydrogenases with different specificities (related to short-chai alcohol dehydrogenases) SEQ ID N ° 699 XAA-PRO DIPEPTIDASE (EC 3.4.13.9)
• Small subunit PAB0068; activator 1, replication factor C, small subunit (RFcS).
• Sequences - the amino acid sequence SEQ ID No. 801 represents the complete sequence (including inteins) of the small subunit;
• amino acid sequence SEQ ID No. 802 represents the sequence of the small subunit in which the two sequences of inteins have been excised; - the amino acid sequences SEQ ID No. 803 and SEQ ID No. 804 respectively represent the two sequences of excised inteins of the sequence SEQ ID No. 802;
• amino acid sequence SEQ ID No. 805 represents the sequence of the large subunit
• sequence of ORF 802 thus corresponds to the sequence of ORF 801, identified above, in which the sequences coding for the intein peptides of sequences SEQ ID NO:
• sequences of ORF 803 and ORF 804 correspond respectively to the sequences coding for the intein peptides of sequences SEQ ID No. 803 and N "804 of ORF 801.
• Initiating site (Nde I): 5'-GGCCTCTAATTTCATATGCGTGACATGGAG-3 'Recovery site, intein 1: direct direction: 5'-GGCCCCCAGGAGTTGGAAAGACAACCGCCGCTCTGGCCCT-3' reverse direction: 5'-AGGGCCAGAGCGGCGGTTGTCCTGTCCTCCT
• Terminator site (BamHI): 5'-GGGGGATCCCTTTATTTCTTCTTTCCGATT-3 '
• PCR is carried out with the following primers: Primers used:
• Terminator site (BamHI): 5 '-GGCCCTACTGGATCCAGCTACTTCTTTATG-3'.
• Antibody A mouse monoclonal against the DNA binding domain.
• Antibody B rabbit polyclonal against the PCNA binding domain; recognizes, in eukaryotes, the region of amino acids 478 to 712 of the large subunit of the RFC.
• TABLE 3 Percentage identity of RFC sequences, small subunit (S) or large subunit (L) between different species of Euryarchaeotes and Crunarchaéotes
• PCNA Nuclear cell proliferation antigen
• Genome reference of Pyrococcus abyssi strain Orsay PAB1465; cell proliferation nuclear antigen (pol 30).
• Gene family [9.1] DNA metabolism / replication, recombination and DNA repair.
• the PCR is carried out with the following nucleotide primers: 0 Initiating site (Nde I): 5 '-AGGTGCAAACATATGCCATTCGAGATAGTC-3'
• Terminator site (Bgl II): 5 '-AGTTAAAGATCTTTACTCCTCAACCCTGGG-3'.
• Vector pBS / SK- Strain: E. coli DH5 ⁇
• Buffer 50 mM Tris-HCl pH 8.8, 1 mM dithiothreitol, 10 mM KC1, 5 mM MgCl 2 , 0.4 mg / ml BSA.
• 1 unit of DNA polymerase is the quantity of enzyme which allows the incorporation of 1 nmole of dNTP per minute, under these experimental conditions.
• YEAST yeast Saccharomyces cerevisiae 258 28916

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