WO2001081580A1

WO2001081580A1 - Origin of replication in the hyperthermophilic pyrococcus archea and its applications

Info

Publication number: WO2001081580A1
Application number: PCT/IB2001/000874
Authority: WO
Inventors: Hannu Myllykallio; Patrick Forterre; Philippe Lopez; Hervé PHILIPPE; Fujihiko Matsunaga
Original assignee: Centre National De La Recherche Scientifique (Cnrs)
Priority date: 2000-04-20
Filing date: 2001-04-20
Publication date: 2001-11-01
Also published as: AU5658401A

Abstract

The present invention relates to an isolated nucleic acid comprising the sequence SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 encoding the origin of replication of Pyrococcus abyssi, Pyrococcus furiosus and Pyrococcus horikoshii respectively, a fragment thereof comprising the minimal genetic region encoding said origin replication functional, a nucleic acid capable of exerting a function of origin of replication and having at least 70 % identity degree with the sequence SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, or a complementary sequence thereof.

Description

"ORIGIN OF REPLICATION IN THE HYPERTHERMOPHILIC PYROCOCCUS ARCHEA AND ITS APPLICATIONS".

FIELD OF THE INVENTION This invention relates to isolated nucleic acids comprising the sequence encoding the origin of replication of Pyrococcus species, vectors comprising said nucleic acids and their use in recombinant DNA technology. The invention is also directed to method for identifying compounds which interact with said nucleic and methods for screening or biosynthetizing organic compound, or for bioremediation comprising the use of host cells, particularly Pyrococcus Archaea, transformed with vectors of the present invention.

BACKGROUND OF THE INVENTION

Despite a rapid expansion in the amount of available archaeal sequence information, little is known about duplication of genetic material in the third domain of life. Recently, comparative genomics has revealed that most archaeal informational processes are similar to those in eukaryotes (1). This is especially striking in the case of DNA replication as all putative archaeal DNA replication proteins have eukaryotic homologs only or, alternatively, are more closely related to their eukaryotic counterparts than to their bacterial ones (2,3). The lack of conservation of replication factors in Eukarya/ Archaea and Bacteria is so striking that their independent development after divergence from a last universal ancestor carrying an RNA genome has been suggested (3). Although recent experimental studies have confirmed predicted enzymatic activities for many open reading frames suggested to encode archaeal replication proteins (4), our understanding of how chromosomes are replicated by these proteins has remained enigmatic. Even whether Archaea have a single replication origin, like Bacteria, or multiple origins, like Eukarya, has been unknown. In some bacteria, GC skew can be used to detect replication origins and termini due to an excess of G over C in the leading strand of replication (5). Conventional GC skew analyses failed to identify strand asymmetry in completely sequenced archaeal genomes (6), suggesting the possibility of multiple origins, in agreement with the eukaryotic nature of the archaeal replication apparatus. However, a single replication origin was suggested by cumulative diagrams of GC or tetramer skews in the Archaea Methanobacterium thermoautotrophicum (7,8) and Pyrococcus horikoshii (8).

SUMMARY OF THE INVENTION

Recognizing the need to provide the origin of replication of Pyrococcus species, particularly of Pyrococcus abyssii, for the development of vector systems which would provide the capability of replication of recombinant vectors in Pyrococcus species, the present inventors have identified the genomic fragment of Pyrococcus abyssii, Pyrococcus furiosus and Pyrococcus horikoshii encoding their origin of replication.

A single origin of bi-directional replication in said Pyrococcus species has been identified by means of cumulative oligomer-skew in silico analyses and identification of an early replicating chromosomal segment. The replication origin in three Pyrococcus species was found to be highly conserved, and several eukaryotic-like DNA replication genes were clustered around it. As in Bacteria, the chromosomal region containing the replication terminus was a hot spot of genome shuffling. Thus, although bacterial and archaeal replication proteins differ profoundly, they are used to replicate chromosomes in a similar manner in both prokaryotic domains.

So, the present invention is directed to an isolated nucleic acid comprising the sequence SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 encoding the origin of replication of Pyrococcus abyssi, Pyrococcus furiosus and Pyrococcus horikoshii.

The invention further concerns an expression, cloning or shuttle vector, such as a plasmid comprising said nucleic acid encoding said origin of replication of Pyrococcus species according to the present invention. The invention further comprises an host cell transformed with said vector according to the invention.

In another object, the present invention is directed to a method for producing a heterologous peptide of interest in Pyrococcus Archaea, comprising transforming said Pyrococcus Archaea with a vector according to the invention, culturing said transformed Pyrococcus Archaea in a suitable culture medium and recovering said target peptide of interest.

The present invention is also directed to a method for identifying compounds which interact with and inhibit or activate said Pyrococcus species origin of replication polynucleotide activity comprising particularly the use of the nucleic acid of the invention.

The present invention further concerns methods for the bioremediation or biosynthetizing organic compound, comprising the use of host transformed cell according to the invention, particularly Pyrococcus species.

Finally, the present invention comprises a method for screening organic compounds capable of being biotransformed by said host cell according to the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A and IB. Panel A: Pyrococcus genomes (PHO, P. horikoshii; PAB, P. abyssi) were compared using a BLAST tool and plotted against each other. Each data point represents 100 nucleotides with more than 80% identity between two genomes. Scale is given in increments of 10⁶ base pairs. Panel B: Non-cumulative and cumulative skews of tetramer GGGT for the two Pyrococcus genomes (abbreviations are as in the panel A). In each graph, abscissa represents the whole length of the genome and is directly comparable to those in the panel A. The skews are defined as the relative excess of word GGGT over its reverse complement ACCC in a sliding window of l/50th of genome. The position of the window is incremented by l/240^th of genome, yielding 240 values. Obtained values are displayed directly as shown on the left side of the panel (non-cumulative skew). The values were also integrated from the start of the genome, positive and negative values resulting in an ascending and a descending slope, respectively. As expected, cumulative diagrams provide a much more convenient and accurate display of the trends of the skew. In the genome of P. hojikoshii and P. abyssi, a well-defined singularity point is detected (indicated by the arrow). Figure 2. ³H-uracil incorporation into alkaline resistant form (DNA) of P. abyssi in the absence (control) and the presence of puromycin (200 μg/ml) is shown. Control experiments (data not shown) using alkaline lysis and DNAse I treatments of isolated nucleic acids from labeled cultures indicated that 30 - 35 % of label was incorporated into DNA. Incorporation of radioactive uracil was measured after TCA precipitation of whole cells by scintillation counting. Figure 3.

Relative labeling intensities for P. abyssi Notl macrorestriction fragments are given. Abscissa indicates the middle point of the different fragments (indicated by roman numerals on the top of the figure) in sequence coordinates. The relative intensity of each fragment is plotted for three different time points of radio-active labeling, as well as using fluorescent detection without puromycin treatment. All curves have a minimal value of 1.0 but have been off-set by increments of 0.25 for a better readability. The location of the well-defined singularity of the Fig. 1 is marked by the arrow. Figure 4.

A physical map of P. abyssi origin region is shown. Notice the presence of genes for an archaeal initiator (Orcl/Cdc6), two subunits of DNA polymerase (DNA Pol), DNA polymerase processivity factor (RF-C), a Rad51 homolog, and putative DNA helicase in the vicinity of the P. abyssi oriC. Figure 5.

The sequence alignment of the upstream regions for P. abyssi, P. furiosus and P. horikoshii cdcβ genes is shown. Nucleotide coordinates 123489-122660, 111568-110772 and 15408- 16230 of P. abyssi, P. horikoshii and P. furiosus, respectively, were aligned with the ClustalW program. Various repeats (shown by arrows with + and - signs referring to their sense strand) as well as AT-rich elements are well conserved between all these organisms. The underlined ATG codon and the arrow underneath of the alignment indicate the first codon of the Cdc6 and the mapped transcriptional start site of P. furiosus cdcό, respectively. Figure 6: Physical map of a P. abyssi chromosomal segment (sequence coordinates 118 000 - 128 000) carrying the predicted replication origin (Myllykallio et al., 2000). Locations of several restriction sites (H, Hindlll; N, Nhel; E, EcoBI; X, Xbaϊ), probes used for N/N 2D analyses as well as a location of the oriC plasmid insert are indicated. Fragments A (4.9 kb Nhel fragment), B ( 5.7 kb EcøRI fragment), C (4.1 kb Hindm fragment), and D (5.6 Xbal fragment) correspond to those analyzed in Figure 7. The central third of the each fragment [a "bubble" detection zone (Linskens and Huberman, 1990)] is indicated by gray shading. A 1.2 kb boxed region upstream of, and partially overlapping with, the P. abyssi cdc6 indicates the region containing an active replication origin as demonstrated in Figure 7. Figure 7: Two-dimensional N N gel analyses of replication intermediates from the predicted replication origin region of P. abyssi. Replication intermediates were prepared from asynchronous P. abyssi cultures using an agarose plug method. Panels A through D correspond to the analysis of restriction fragments A to D indicated in Figure 6, while panel E shows the expected pattern for fragments containing either one replication fork ("Y" arc), or two replication forks with an internal initiation site ("bubble" arc). The filled arrows indicate the bubble arcs observed for fragments B and C by probe 2 (similar results were obtained when probe 1 was used). In addition to a well defined Y arc, fragments A and D show weak signals (small arrows) detected by probe 1. These signals are consistent with an asymmetrically located replication origin within the central third of the fragments B and C. The mobilities of the dark spots on the line traced by linear fragments are due to impartial digests.

Figure 8: Transient transformation assay using a P. abyssi oriC plasmid. Panel A: Schematic representation of a Dpnl assay modified according to DePamphilis, 1995. Panel B: Conversion of Dpnl sensitive P. abyssi oriC plasmid preparation isolated from a dam+ E. coli strain into Dpnl resistant forms by DNA replication in P. abyssi. "Control" refers to a control transformation reaction performed with a plasmid lacking the oriC insert. Plasmids were detected using a 2.9 kb probe corresponding to an entire pBluescript cloning vector. Panel C: Structure of the oriC region with the identification of the two duplex unwinding elements (DUE) and conserved nucleotide repeats indicated by arrows. DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention concerns an isolated nucleic acid comprising the sequence SEQ ED NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 encoding the origin of replication of Pyrococcus abyssi, Pyrococcus furiosus and Pyrococcus horikoshii respectively, a fragment thereof comprising the minimal functional portion of said sequence SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, a nucleic acid capable of exerting a function of origin of replication and having at least 70 %, preferably 80 %, 85 %, 90 %, 95 % and 99 %, identity degree with the sequence SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, or a complementary sequence thereof. The sequence SEQ ID NO: 1 is the first nucleic sequence which is depicted in figure

5 (line "P. abyssi") and containing 824 bp.

The sequence SEQ ID NO: 2 is the second nucleic sequence which is depicted in figure 5 (line "P. furiosus") and containing 831 bp.

The sequence SEQ ID NO: 3 is the third nucleic* sequence which is depicted in figure 5 (line "P. horikoshii") and containing 851 bp.

It should be understood that the present invention does not relate to the genomic nucleotide sequences in their natural chromosomal environment, that is to say in the natural state. They are sequences which have been isolated, that is to say that they have been collected directly or indirectly, for example by copying, their environment having been at least partially modified.

Thus, these sequences may also be nucleic acid which have been partially modified or carried by sequences which are at least partially different from the sequences carrying them naturally.

The term "degree or percentage of sequence identity" refers to degree or percentage of sequence identity between two sequences after optimal alignment as defined in the present application.

Two amino-acids or nucleotidic sequences are said to be "identical" if the sequence of amino-acids or nucleotidic residues, in the two sequences is the same when aligned for maximum correspondence as described below. Sequence comparisons between two (or more) peptides or polynucleotides are typically performed by comparing sequences of two optimally aligned sequences over a segment or "comparison window" to identify and compare local regions of sequence similarity. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman,. Ad. App. Math 2: 482 (1981), by the homology alignment algorithm of Neddleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. (U.S.A.) 85:2444 (1988), by computerized implementation of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), or by visual inspection.

"Percentage of sequence identity" (or degree or identity) is determined by comparing two optimally aligned sequences over a comparison window, where the portion of the peptide or polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical amino-acid residue or nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

The definition of sequence identity given above is the definition that would use one of skill in the art. The definition by itself does not need the help of any algorithm, said algorithms being helpful only to achieve the optimal alignments of sequences, rather than the calculation of sequence identity. From the definition given above, it follows that there is a well defined and only one value for the sequence identity between two compared sequences which value corresponds to the value obtained for the best or optimal alignment.

In the BLAST N or BLAST P " BLAST 2 sequence" (Tatusova et al, "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol. Lett. 174 : 247-250) software which is available in the web site http://www.ncbi.nlm.nih.gov/gorf/bl2.html and habitually used by the inventors and in general by the skilled man for comparing and determining the identity between two sequences. The "open gap penaltie" and « extension gap penaltie » parameters which depends on the substitution matrix selected regarding the nature and the length of the sequence to be compared is directly selected by the software (i.e. "5" and "2" respectively for substitution matrix BLOSUM-62). The identity percentage between the two sequences to be compared is directly calculated by the software.

The term "fragment comprising the minimal functional portion of sequence SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 refers to a fragment having at least 20, 30, 40, 50,

75, 100, 150, 200, 250 and 300 consecutive nucleotides of the sequence SEQ ID NO: 1,

SEQ ID NO: 2 or SEQ ID NO: 3, and which is capable of exerting partially or totally the function normally associated to an origin of replication in a micro-organism.

The invention further concerns a vector which comprises the nucleic acid according to the invention.

In a preferred embodiment, said vector according to the invention further contains a nucleic acid encoding the Cdc6/orcl protein of Pyrococcus species, particularly of Pyrococcus abyssi whose nucleic sequence is avalaible at web site www.genoscope.cns.fr/cgi-bin/Pab.cgi under the sequence reference PAB2265 (orcl), and corresponding to the nucleic acid beginning at position 122700 and stopping at position 121405 of the complete genomic sequence of Pyrococcus abyssi.

The invention further concerns a vector which comprises the nucleic acid having the sequence corresponding to the position nt 120687-124803 in the genomic sequence of Pyrococcus abyssi (available at the above-cited genoscope web site) or the corresponding nucleic acid of Pyrococcus furiosus or horikoshii.

In a particularly embodiment, the present invention concerns a vector according to the invention which comprises a second nucleic acid encoding an origin of replication functional in at least one selected from the group consisting of bacteria, such as Escherichia coli or Bacillus subtilis, yeast, such as Saccharomyces cerevisiae, or mammalian cells. Such vector can be used for the preparation of shuttle vector.

In a preferred embodiment, the present invention relates to vectors according to the invention, further comprising a gene insert foreign to the transformed host cell. By "a gene insert foreign to the transformed host cell" is meant a gene encoding a target heterologous peptide of interest which is desired to express in a host cell. The target peptide will be"heterologous" if it is encoded by a DNA sequence that is foreign, i.e., originates from a donor different from the host or is a chemically synthesized gene, and can include a donor of a different species from the host. The heterologous gene codes for a peptide ordinarily not produced by the host organism.

The target peptide may be encoded by a structural gene, in which case a mature peptide product would be expressed.

Alternatively, the target peptide may comprise the prepro-, pro- or pre- forms of the peptide. The present invention further concerns an expression, cloning or shuttle vector containing a nucleic acid sequence according to the invention.

In a preferred embodiment, the shuttle vector according to the invention, is an E. coli/Pyrococcus species shuttle vector, particularly an E. coli/Pyrococcus abyssi shuttle vector. The vectors according to the invention, characterized in that they comprise the elements allowing the expression and/or the secretion of said target peptide in a host cell, also form part of the invention.

The said vectors will preferably comprise a promoter, signals for initiation and termination of translation, as well as appropriate regions for regulation of transcription. They must be able to be stably maintained in the cell and may optionally possess particular signals specifying the secretion of the translated protein.

These different control signals are chosen according to the cellular host used. To this end, the nucleic acid sequences according to the invention may be inserted into autonomously replicating vectors inside the chosen host. Among the autonomously replicating systems, there will be preferably used according to the host cell, systems of the plasmid or viral type. Persons skilled in the art know the technologies which can be used for each of these systems.

Such vectors will be prepared according to the methods commonly used by persons skilled in the art, and the clones resulting therefrom may be introduced into an appropriate host by standard methods such as, for example, lipofection, electroporation or heat shock.

The invention also includes a method for the preparation of a shuttle vector, comprising including in the nucleic sequence coding for the origin of replication of Pyrococcus species, a second nucleic sequence which permits direct control of replication at the second origin.

Methods for the preparation of said shuttle vectors suitably comprise ligating a first nucleic acid coding for first origin of replication with a second nucleic acid coding for the secondary replication system. The nucleic acid which permits direct control of replication at the second origin may be incorporated into the second nucleic acid either before or after ligation with the first nucleic acid.

The present invention is also directed to host cell transformed with a vector according to the invention.

In a preferred embodiment, said host cell is a Pyrococcus genus Archaea.

In a more preferred embodiment, said host cell is Pyrococcus abyssi, Pyrococcus furiosus or Pyrococcus horikoshii.

These cells may be obtained by introducing into the host cells a nucleotide sequence according to the invention inserted into a vector as defined above, and then culturing the said cells under conditions allowing the replication and/or expression of the transfected nucleotide sequence. These cells can be used in a method for the production of a recombinant polypeptide and can also serve as a model for analysis and screening.

Said method for the production of a polypeptide in recombinant form is itself included in the present invention, and is characterized in that the transformed cells according to the invention are cultured under conditions allowing the expression of a recombinant polypeptide, and in that the said recombinant polypeptide is recovered.

Thus, in another aspect, the invention is directed to a method for producing a heterologous target peptide in Pyrococcus Archaea, comprising: a) transforming said Pyrococcus Archaea with a vector according to the invention; b) culturing the thus transformed Pyrococcus Archaea in a suitable culture medium; and c) recovering said target peptide.

The invention also relates to a method for identifying compounds which interact with and inhibit or activate the function of origin of replication of the nucleic acid according to the invention comprising the step of: a) contacting a composition comprising a nucleic acid according to the invention with the compound to be screened under conditions to permit interaction between the compound and said nucleic acid to assess the interaction of a compound, such interaction being associated with a second component capable of providing a detectable signal in response to the interaction of said nucleic acid with the compound; and b) determining whether the compound interacts with an activates or inhibits the function of origin of replication of said nucleic acid by detecting the presence or absence of a signal generated from the interaction of the compound with said nucleic acid.

The present invention also provides a method for the bioremediation, comprising the use of host cell according to the invention.

The target recombinant polypeptide which can be expressed in the host cells, such as transformed Pyrococcus species, according to the invention, may be polypeptide, such as enzyme or co-enzyme, capable of abolishing or reducing the toxicity of exogenous substrates. This target recombinant polypeptide could, for this reason, be used as biocatalyst either for the degradation of environmental pollutants or for the biosynthesis of organic compounds.

In addition, the invention relates to a method for biosynthetizing organic compound, comprising the step of: a) bringing organic substrate of the polypeptide encoded by the gene insert foreign to the transformed host cell into contact with said transformed host cell according to the invention under conditions allowing the synthesis of said organic compound by biotransformation of the substrate by said transformed host cell; and b) recovering said organic compound.

The invention also relates to a method for screening organic compounds capable of being biotransformed by a host cell according to the invention comprising the step of: a) bringing said organic compounds into contact with said host cell under conditions allowing the biotransformation of said organic compound; b) analysing the obtained compounds; and c) selecting the biotransformed organic compounds.

The invention also relates to the use of vectors according to the present invention for improving Pyrococcus strains as expression host cells, particularly by specific inactivation of genes, such as proteases, using anti-sens RNA technology. Having now generally described this invention, the same will become better understood by reference to certain specific examples which are included herein for purposes of illustration only and are not intented to be limiting unless otherwise specified.

EXAMPLES

Example 1

The genome of Pyrococcus abyssi has been recently sequenced (9), allowing the comparison of strand asymmetry in the genomes of two closely related organisms (P. abyssi and P. horikoshii) in which major chromosomal rearrangements have occurred after their divergence (Fig. 1A). Although normal diagrams of oligomer skew (here tetramer GGGT) could suggest multiple origins (Fig. IB), cumulative skew analyses of the same tetramer gave smooth curves for the two genomes with one well-defined singularity and one broad peak, both at similar locations. The cumulative skew pattern thus appears to be a stable feature of genome composition as it has not been altered by the chromosomal rearrangements between the two Pyrococcus species (Fig. 1A). Similar results (data not shown) were obtained when the genome sequence of a third Pyrococcus species, P. furiosus, was analyzed (10). The shape of the cumulative skew diagrams in Fig. IB could be explained a priori by two different replication mechanisms: one of the two singularities could represent a bidirectional replication origin, or they both could represent mono-directional replication origins. To distinguish between these possibilities, we sought to identify an early replicating chromosome segment with information deduced from the complete genome sequence of P. abyssi. In preliminary experiments, we found that uracil, but not thymidine, could be used to label efficiently chromosomal DNA when cells were grown anaerobically at 95°C (11). DNA synthesis in vivo was fully inhibited by puromycin, a universal protein synthesis inhibitor (Fig. 2). By analogy to the bacterial system, the residual incorporation of label into DNA observed in the presence of puromycin suggested that this inhibitor specifically blocks the initiation step of DNA replication in P. abyssi.

In a further experiment, DNA replication was arrested by puromycin, in order to increase the proportion of replication forks located close to the origin. After drug removal, newly replicating DNA was radioactively labeled, and the chromosomal distribution of radioactivity was determined by calculating the relative abundance of labeled Notl fragments of P. abyssi, as resolved by contour-clamped homogeneous electric field electrophoreses (CHEF).

Briefly, P. abyssi cultures were grown using enriched VSM medium to an ODδoo = 0.12, and were treated with puromycin (200 μg/ml) for 110 min. The drug was removed by ^' extensive washes with growth medium at + 4 °C. Treated cells were then re-inoculated to OD_δoo = 0.10 in the presence of ¹⁴C-uracil (2 μCi/ml), and labeling was continued for indicated times. Agarose plugs for CHEF analyses (6.5 V/cm, a linear pulse ramp 3 - 17 s for 20 h, +14 °C) were prepared from 5 ml cultures using a modification of standard protocols. Gels and filters (1-2 weeks exposure to Storage Phosphor screens) were analyzed with a Storm imaging system (Molecular Dynamics). Radioactivity of each Notl fragment was determined using ImageQuant software following the manufacturer's recommendations. To ensure that the results obtained were not an artifact of the puromycin treatment, the relative steady state levels of Notl fragments were also investigated in untreated early exponential stage cultures. In this case, CHEF gels were stained with VistraGreen (Molecular Dynamics), a D A specific fluorescence dye, and were directly scanned using a blue fluorescence mode of a Storm system. Relative frequencies for fragments were obtained by dividing the amount of radioactivity/fluorescence of a given band by its size calculated based on the determined genome sequence. All signals analyzed were within a linear range of a Storm system. Values given were normalized to the minimal value of one arbitrary unit, and are averages of two independent measurements.

After 30 minutes of labeling, the highest relative intensity was reproducibly observed for the 80 kb fragment (Fragment II in Fig. 3) corresponding to the well-defined singularity present in skew diagrams (marked with the arrow in Figs. 1 and 3). The lowest value was obtained for the fragment located opposite of the fragment II on the P. abyssi genome, corresponding to the broad peak of the cumulative skew diagram. As expected for an early replicating fragment, the relative labeling intensity of the fragment II gradually decreased when labeling was continued for up to 90 min. The simplest explanation for our in silico and experimental data is thus that Pyrococcus sp. chromosomes are replicated bi- directionally from a fixed single origin located within the 80 kb fragment described above, and that the replication terminus is located approximately opposite of the origin. Finally, the experimentally mapped replication origin of P. abyssi corresponds to a transition from negative to positive GC skew, indicating an excess of G over C in the leading strand of these Archaea (5).

The archaeal replication origin region we identified does not contain typical sequences recognized by the bacterial initiator protein nor is DnaA obvious in archaeal genomes (2). Instead, they all contain an archaeal homolog of Orcl/Cdc6 proteins that are involved in the initiation step of DΝA replication in eukaryotes (11). These archaeal Orcl/Cdc6 homologs are located immediately downstream of a large intergenic sequence (about 800 bp) (13). This region is interrupted by several stop codons in the three Pyrococcus genomes indicating that it does not contain genes that could have been missed during the genome annotation.

Strikingly, this is the only highly conserved intergenic sequence of more than 600 bp to be conserved in the three Pyrococcus genomes (14) suggesting a strong functional constraint for this region. Indeed the genome of P. abyssi contains 38 intergenic regions longer than 600 bp. When run through the BLAST program, only 7 of these yield to an expect score lower than le-10 against P. horikoshii genome while the origin region gets an expect score of le-33, the use of the TBLASTX program indicates that the high scores of the remaining 6 intergenic regions might be due to some hard-to-identify ORFs. This sequence contains a 45 bp long central core with a single nucleotide substitution between all three species, as well as many conserved direct and inverted repeats, similar to those present in the predicted origin of M. thermoautotrophicum ((8), see also Fig. 5). In the three Pyrococcus species, the described origin region carries many genes encoding orthologs of proteins involved in both the initiation (e.g. Cdc6/Orcl) and the elongation steps of eukaryotic DNA replication (e.g. RF-C) (Fig. 4). Grouping of replication genes near the origin has been reported in some bacteria and possibly helps the effective assembly of replication forks onto the origin (12).

Whereas the replication origin is highly conserved between the three Pyrococcus species, the chromosomal region containing the replication terminus is a hot spot of genome shuffling as shown by the genome to genome comparison between P. abyssi and P. horikoshii (Fig. 1A). Genetic instability in the terminus region has been also observed for Bacteria (13), and it could reflect a common mechanism for the termination of replication and/or chromosome segregation in all prokaryotes. Indeed, in addition to bacterial-like cell division genes already detected in Archaea [e.g. FtsZ, MinD and SpoJ (8)] the inventors noticed that Pyrococcus sp. and other Archaea contain a XerC and XerD homolog (PAB0255 in P. abyssi), which in Bacteria are involved in the resolution of chromosomes at the end of the replication process (14).

The successful identification of an archaeal replication origin validates the use of archaeal replication proteins such as Cdc6/Orcl and MCM to understand the functioning of their eukaryotic homologs (15). Since the genome of P. abyssi encodes only two putative initiator proteins (MCM and Cdc6/Orcl) to be compared to a complex set of proteins involved in yeast replication initiation, the archaeal in vitro replication system could represent a minimal eukaryo tic-like mechanism for initiation of DNA replication. By itself, the replication of a prokaryotic chromosome from a single bi-directional replication origin by an eukaryotic-like machinery raises fundamental questions. For example, inventors' data indicate that the P. abyssi chromosome is replicated in approximately 45 minutes, i.e. once during every cell division. In this species each replication fork must then be traveling (20 kb/min along the archaeal chromosome. This value is much higher than those observed for Eukarya (2-3 kb/min), while similar values have been obtained for Bacteria [E. coli, 50 kb/min; Caulobacter crescentus; 21 kb/min (16)]. The faster replication rate in Archaea than in Eukarya could reflect the differences between the two replication apparatus [e.g. different replicative DNA polymerase and streamlining of several factors (2)] or a less compact nucleosomal structure (as expected for an actively transcribed genome with a high coding capacity).

The inventors failed to identify strand asymmetry by cumulative skew or tetramer analyses in the genomes of Methanococcus jannashii, Archaeoglobus fulgidus and

Aeropyrum pernix and in some Bacteria (17). Whether this implies the presence of multiple replication origins in these organisms or a limitation of the cumulative skew method remains to be established. However, the identification of both eukaryal and bacterial features in the Pyrococcus DNA replication mechanism already has evolutionary implications. The marked differences between replication factors in Eukarya/ Archaea and Bacteria have been explained either by their high evolutionary rate triggered by the different replication modes in Eukarya and Bacteria (1), an independent development after their divergence (3), or a massive non-orthologous displacement (18). Since in the case of P. abyssi eukaryotic-like replication proteins are utilized in the manner similar to Bacteria, the first possibility is unlikely. It remains now to be established if similarities of archaeal and bacterial replication systems are a consequence of their common prokaryotic lifestyle (convergence) or if they are evolutionary related (homologous). The latter hypothesis is supported by the observation that DnaA and Cdc6 belong to the same protein superfamily (19), suggesting that all cellular replication initiation mechanisms originated in a DNA- based ancestor.

Example 2

2.1. Materials and methods

2.1.1. Cell culture techniques

P. abyssi GE5 (strain Orsay) and GE9, devoid of any plasmids, were cultivated anaerobically in liquid medium at 95 °C using enriched VSM medium as described earlier (20). Cultures were inoculated with l/20th of the final culture volume using freshly obtained inoculate. Cells were harvested after 5-6 hours of growth at approximate cell densities of 5x10⁷ (for N/N 2-D gel electrophoresis) or l-2xl0⁷ (for other purposes) cells/ml. The absence of chromosomal rearrangements in the P. abyssi genome was confirmed by contour-clamped homogenous electric field electrophoreses (data not shown).

2.1.2. Neutral/neutral two-dimensional agarose gel electrophoresis

P. abyssi cells from asynchronous cultures were washed and re-suspended under aerobic conditions using buffer A (250 mM NaCl; 25 mM Tris-Cl, pH 8.0; 10 mM EDTA). Cells were suspended at 10¹⁰ cells/ml, and were encapsulated using an equal volume of 0.8 % low gelling temperature agarose (Type VII, Sigma) in buffer A. DNA was prepared from agarose plugs after their overnight treatment in 1 mg ml proteinase K; 10 mM Tris-Cl, pH 9.1; 0.5 M EDTA at 37 °C. Obtained plugs, which were washed extensively with buffer A and TE prior to further manipulations, contained approximately 20 - 30 μg of DNA/ ml.

Intact DNA was digested in agarose plugs using the indicated restriction enzymes following manufacturers recommendations for pulse field electrophoresis qualified restriction enzymes (Promega or New England Biolabs), including 2 units of agarase (Sigma) per 100 μl plug. After completing the digestions, the plugs were extracted with phenol and chloroform followed by isopropanol precipitation of the DNA samples and two washes using 70 % ethanol at room temperature. 10 - 15 μg of digested DNA were run on neutral/neutral 2-D gels (21) using lxTBE buffer in cold room [1st dimension: 0.4 % agarose, 0.7 V/cm, ~ 40 hours; 2nd dimension: 1 % agarose, 3 V/cm, 13 - 15 hours]. Southern blots using Hybond-N (Amersham) filters and probe preparation were performed as described (22). All radioactive signals were revealed by exposing the filters to Phospholmager screens for up to 72 hours, and signals were analyzed using the ImageQuant software (Molecular Dynamics). 2.1.3. Transformation of P. abyssi

The upstream region of cdcό [(Figure 6); P. abyssi sequence coordinates 122628 - 123463] was amplified by Pfu DNA polymerase using genomic DNA as template with appropriate restriction sites included in primer sequences. Plasmid pFM200 (P. abyssi oriC plasmid) was obtained by cloning the obtained 0.8 kb PCR fragment into HindΩI and Bam R sites of pBluescriptll KS+. All plasmid constructs were maintained inE. coli d m+ SURE strain (Stratagene). For transformation, 500 ng of circular ρFM200 DNA was combined with P. abyssi GE9 spheroplasts in the presence of 12.5% PEG- 1000 (Lucas et al, unpublished data). After transformation, the cells were allowed to divide up to 5 times in enriched medium without phenotypic selection. Plasmid populations from harvested P. abyssi cells were isolated using Trizol (GibcoBRL) DNA extraction reagent following manufacturers recommendations. Dpnl assays were performed as described earlier (23) after confirming the absence of any material possibly inhibiting restriction endonucleases in the DNA preparations used. Data shown is representative of three independent transformation assays.

2.2. Results

2.2.1. An active replication origin is located in the vicinity of the P. abyssi cdcδ initiator gene. As earlier inventors observations have suggested a physical linkage between a replication origin and the P. abyssi cdcό gene, the inventors investigated how a 10 kb chromosomal segment centered at the cdcό gene (P. abyssi sequence coordinates 118 000 to 128 000; Figure 6) is replicated in vivo. A replication pattern of this region was revealed using neutral/neutral two-dimensional (N/N 2-D) agarose gel electrophoreses (24), which rely on electrophoretic separation of the different classes of replication intermediates (Figure 7E). Total intact DNA, thus including all replication intermediates, was gently prepared from P. abyssi exponential growth phase cells embedded in agarose plugs. After N/N 2-D gel electrophoreses, the replication mode of four restriction fragments covering the aforementioned P. abyssi chromosomal region was investigated using two specific radioactive probes (Figure 6). These analyses (Figure 7) indicated that the fragments A and D, which are distally located to the cdcό [4.9 kb Nhel fragment (Panel A); 5.6 kb Xbal fragment (Panel D)], produced a strong "Y" arc; hence, they are replicated with one fork travelling from one end to the other. In contrast, the 5.7 kb EcόRI (Panel B) and 4.1 kb HindlU (Panel C) fragments produced a composite pattern consisting of a well defined "bubble" arc (indicated by filled arrows) and a typical "Y" arc. As "bubble" arcs can be detected reliably only when a replication origin is located in the central third of a restriction fragment (25) and cdc6 is centrally located only within the latter fragments, these results demonstrate that the P. abyssi chromosomal replication origin is located in a 1.2 kb window (indicated by a box in Figure 6) either just upstream, or partially overlapping, the cdc6 locus. Inventors data also indicate that the P. abyssi origin studied here fires symmetrically since the inventors were unable to detect any obvious replication pause sites within this 10 kb chromosomal region.

As inventors earlier data have shown that there is a single bi-directional replication origin in the P. abyssi chromosome (20), it is unclear why P. abyssi oriC shows a composite pattern in these experiments. The simplest explanation for this observation is that the replication bubbles become nicked during the sample preparation, thus changing their electrophoretic mobility. A similar composite pattern in N/N 2-D gel data was previously observed for the single chromosomal replication origin of Mycoplasma capricolum (26). Therefore, analogous composite patterns observed for replication origins in Bacteria and Archaea might be somehow related to the much higher replicon sizes and/or replication speed in prokaryotes compared to Eukarya. 2.2.2. Dpnl assays indicate that the 0.8 kb intergenic region upstream of the cdcό is essential for in vivo replication

To determine if the mapped origin is located within the intergenic region upstream of cdcό and whether it can confer autonomous replication in vivo, a plasmid (pFM200) carrying this 0.8 kb intergenic region was constructed. To detect in vivo replication of this plasmid, the inventors benefited from their recent experimental observation that, in contrast to E. coli, P. abyssi lacks a functional deoxyadenosine methylation (dam) system (F.M. and H.M., unpublished observation). When plasmid pFM200 was isolated from a dam+ E. coli strain, all of its 15 recognition sites for Dpnl restriction endonuclease (GATC) were methylated by dam methylase. Upon transformation of a methylated pFM200 plasmid preparation into P. abyssi lacking this methylation system, demethylation of transforming plasmid DNA is brought about by its active replication in P. abyssi. The methylation state of plasmid pools isolated from transiently transformed P. abyssi strain GE9 was thus monitored using Dpnl restriction endonuclease that cuts its recognition sequence differentially depending on the methylation status of the isolated DNA (Figure 8 A). As shown in Figure 8B, the pFM200 plasmid was still present in transformed P. abyssi cells after several cell divisions in the absence of phenotypic selection. Moreover, a sub- population of oriC plasmid was converted into a Dpnl resistant form. On the contrary, the control plasmid lacking the insert was not detected after transformation, possibly reflecting instability of non-replicating DNA at the high temperature.

This region contains several copies of a direct repeat (Figure 8C), which can be further divided into two partially symmetric half-sites. Two additional longer repeats, earlier unnoticed, are also present within this replication origin (indicated by long arrows in Figure 8C). In addition, mimicking physiological growth conditions of P. abyssi (95 °C and 500 mM NaCl), the inventors calculated DNA helical stability from the base sequence for this intergenic region in the circular chromosomal DNA according to the method described earlier (27). As shown in Figure 8C, these analyses detected two putative DNA unwinding elements (DUE) located approximately 600 and 400 nucleotides 5' upstream of a start codon for the P. abyssi Cdcό. The longer one of these unwinding elements corresponds to an AT-rich region reported earlier (20).

Jacob et al (1964) (28) proposed in their replicon model that in bacteria, a transacting factor activates a cis-acting DNA element, the replicator, to initiate DNA replication at a specific chromosomal locus. Inventors analyses of P. abyssi DNA replication in vivo now extend this model to the third domain of life, the Archaea, by identifying for the first time an archaeal chromosomal replication origin (oriC). The inventors have shown by N/N 2-D gel electrophoreses (Figure 7) that a single active replication origin, physically linked to the cdcό gene, is present in the P. abyssi chromosomal region. In addition, using transient transformation assays (Figure 8), the inventors have provided the first evidence that cis-acting elements necessary for the P. abyssi replication initiation are located within the 0.8 kb region immediately upstream of P. abyssi cdcό (Figure 8). Thus, initiation sequences in P. abyssi are non-random and well defined.

REFERENCES

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7. A. Grigoriev, Nucleic Acids Res. 26, 2286 (1998). In the case of Mb. thermoautotrophicum, a single replication origin has been also predicted using an independent method [E.P.C. Rocha, A. Danchin, A. Viari, Mol Microbiol 32, 11 (1999)].

8. P. Lopez, H. Myllykallio, H. Philippe, P. Forterre, Mol Microbiol . 32, 883 (1999).

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J.P.J Chong, M.K. Hayashi, M.N. Simon, R-M. Xu, B. Stillman, Proc. Nat Acad. Sci.

U.S.A., 97, 1530 (2000). 16. B. Stillman in DNA replication in eukaryotic cells, M. DePamphilis Ed. (Cold Spring

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Sci. USA 86, 119 (1989).

17. P. Lopez, H. Philippe, unpublished data.

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21. Brewer B., and Fangman, W.L. (1987) S. cerevisiae. Ce// 51, 463-471. 22. Shinomiya, T., and Ina, S. (1991) Nucleic Acids Res. 19, 3935-3941.

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25. Linskens, M.H., and Huberman, J.A. (1990) Ambiguities in results obtained with 2D gel replicon mapping techniques. Nucleic Acids Res. 18, 647-652. 26. Miyata, M., and Fukumura, T. (1997) Asymmetrical progression of replication forks just after initiation on Mycoplasma capricolum chromosome revealed by two-dimensional gel electrophoresis. Gene 193, 39-47.

27. Natale, D.A., Schubert, A.E., and Kowalski, D. (1992) DNA helical stability accounts for mutational defects in a yeast replication origin. Proc. Natl. Acad. Sci. USA 89, 2654- 2658.

28. Jacob, F., Brenner, S., and Cuzin, F. (1964) On the regulation of DNA replication in Bacteria. Cold Spring Harbor Symp. Quant. Biol. 28, 329-348.

Claims

WHAT IS CLAIMED IS:

1. An isolated nucleic acid comprising the sequence SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 encoding the origin of replication of Pyrococcus abyssi, Pyrococcus furiosus and Pyrococcus horikoshii respectively, a fragment thereof comprising the minimal genetic region encoding said origin replication functional, a nucleic acid capable of exerting a function of origin of replication and having at least 70 % identity degree with the sequence SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, or a complementary sequence thereof.

2. A vector which comprises the nucleic acid according to claim 1.

3. The vector according to claim 2 comprising in addition a nucleic acid encoding the Cdc6/orcl protein of Pyrococcus abyssi, Pyrococcus furiosus or Pyrococcus horikoshii.

4. The vector according to claim 1 or 2, wherein said vector is an expression, cloning or shuttle vector.

5. The shuttle vector according to claim 4 which comprises a second nucleic acid encoding an origin of replication functional in at least one selected from the group consisting of bacteria, yeast or mammalian cells.

6. The shuttle vector according to claim 5 wherein said second nucleic acid encoding an origin of replication functional inE.coli.

7. The shuttle vector of claim 6, wherein said vector is an E. coli/ Pyrococcus abyssi shuttle vector.

8. The vector according to anyone of claims 2 to 7, further comprising a gene insert of interest foreign to the transformed host cell.

9. A host cell transformed with a vector according to anyone of claims 2 to 8.

10. The host cell of claim 9 wherein said host cell is a Pyrococcus genus

Archaea.

11. The host of claim 10 wherein said host cell is Pyrococcus abyssi.

12. A method for producing a heterologous target peptide in Pyrococcus species Archaea, comprising: a) transforming said Pyrococcus Archaea with a vector according to anyone of claims 2 to 8; b) culturing the thus transformed Pyrococcus Archaea in a suitable culture medium; and c) recovering said target peptide.

13. A method for identifying compounds which interact with and inhibit or activate function of the origin of replication of Pyrococcus abyssi, Pyrococcus furiosus or Pyrococcus horikoshii comprising the step of: a) contacting a composition comprising a nucleic acid of claim 1 with the compound to be screened under conditions to permit interaction between the compound and said nucleic acid of claim 1 to assess the interaction of a compound, such interaction being associated with a second component capable of providing a detectable signal in response to the interaction of said nucleic acid of claim 1 with the compound; and b) determining whether the compound interacts with and activates or inhibits an activity of said nucleic acid of claim 1 by detecting the presence or absence of a signal generated from the interaction of the compound with said nucleic acid of claim 1.

14. A method for the bioremediation, comprising the use of host cell according to anyone of claims 9 to 11.

15. A method for biosynthetizing organic compound, comprising the step of: a) bringing organic substrate of the polypeptide encoded by the gene insert foreign to the transformed host cell.into contact with said transformed host cell according to anyone of claims 9 to 11 under conditions allowing the synthesis of said organic compound by biotransformation of the substrate by said transformed host cell; and b) recovering said organic compound.

16. A method for screening organic compounds capable of being biotransformed by a host cell according to anyone of claims 9 to 11 comprising the step of: a) bringing said organic compounds into contact with said host cell under conditions allowing the biotransformation of said organic compound; b) analysing the obtained compounds; and c) selecting the biotransformed organic compounds.

17. Use of a vector according to anyone of claims 2 to 8 for improving Pyrococcus species as expression recombinant host cells.

18. Use according to claim 17 wherein the method for improving Pyrococcus species comprises the use of anti-sens acid nucleic for inactivating at least a specific gene of said Pyrococcus species.