EP0705337A1 - Fragment d'adn de clostridium bifermentans portant les genes codant pour des proteines associees a une activite insecticide - Google Patents

Fragment d'adn de clostridium bifermentans portant les genes codant pour des proteines associees a une activite insecticide

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Publication number
EP0705337A1
EP0705337A1 EP94920513A EP94920513A EP0705337A1 EP 0705337 A1 EP0705337 A1 EP 0705337A1 EP 94920513 A EP94920513 A EP 94920513A EP 94920513 A EP94920513 A EP 94920513A EP 0705337 A1 EP0705337 A1 EP 0705337A1
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Prior art keywords
fragment
protein
sequence
polypeptide
nucleotide
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EP94920513A
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German (de)
English (en)
French (fr)
Inventor
Luc Nicolas
Jean-François CHARLES
Armelle Delecluse
Frédérique BARLOY
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Institut Pasteur
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Institut Pasteur
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    • 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
    • C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/50—Isolated enzymes; Isolated proteins

Definitions

  • the invention relates to new toxins isolated from a strain of anaerobic bacteria of the species Clostridium bifermentans.
  • the CH18 strain of Clostridium bifermentans serovar alaysia (Cbir, has been described by H. de Barjac and M. Sebald, in C.R. Acad. Sci. Paris t. 310, Series III, p. 383-387, 1990.
  • the inventors have demonstrated in a bacterium of the species Clostridium bifermentans, the existence of larvicidal activity.
  • the invention provides for the first time toxins present in anaerobic bacteria, capable of having an activity toxic and in particular larvicide towards arthropods, and in particular Diptera type insects.
  • the Clostridium bifermentans seromen malaysia strain used is harmless vis-à-vis the mammals and fish tested as well as vis-à-vis all aquatic organisms apart from the natural target of the larvicidal activity of Cb (Thiery and al. (1992) J. econ. Entomol. 85 (5), 1618-1623).
  • the subject of the invention is therefore nucleotide sequences coding for polypeptides capable of participating in the toxic activity vis-à-vis arthropod larvae and in particular insects, in particular Diptera such as mosquitoes or black flies.
  • the invention also relates to these polypeptides as well as to recombinant cells containing the nucleotide sequences and the nucleotide fragments of the invention, under conditions allowing their expression.
  • antibodies recognizing the polypeptides of the invention as well as compositions with larvicidal activity.
  • oligonucleotide probe 18A TGT GAA GTI AAT TGT GA
  • oligonucleotide probe 16A TGT CAT ATI GAA GCI GTI AAT GAA GG
  • probes 66A ATG AAT ACI AAT ATI TTT TCI ACI AA
  • 66B TC IGG TTC ICC ATA IAT CCA TTC ATC
  • This "target” is generally an arthropod and more particularly an insect, in particular of the Diptera family and for example, a mosquito or blackfly larva.
  • a nucleotide sequence codes for a protein or a polypeptide having the capacity to participate in the toxic activity of the expression products of the 7 kb Xbal fragment contained in the plasmid pCBM1 deposited at the CNCM under the n ° 1-1317, since the deletion or the alteration of this sequence within the said plasmid leads to a reduction or a suppression of the toxic activity with regard to Diptera larvae and in particular of mosquito or blackfly larvae, observed when this plasmid is introduced into a recombinant cell which does not naturally have larvicidal activity, for example a Clostridium bifermentans cell.
  • Other recombinant cells such as those described by Delécluse A. et al (1988) Mol. Gen. Broom. 214: 42-47) can be implemented.
  • the toxic activity can be evaluated by measuring the LC 50 (lethal dose for 50% of the insects or more generally of the "target" on which we want to evaluate the toxic activity).
  • a nucleotide sequence according to the invention can code for a protein, a polypeptide or a peptide necessary for the induction of larvicidal activity or / and can code for a protein, a polypeptide or a peptide influencing the level of expression of the toxicity vis-à-vis a given target.
  • tests carried out with recombinant cells naturally lacking larvicidal activity, transformed by the plasmid pCBM1 under conditions allowing the expression of the genes contained in the inserted DNA fragment can be used as a basis comparison for testing the larvicidal activity of proteins or parts of proteins expressed by a nucleotide sequence according to the invention.
  • protein denotes within the scope of the application, any amino acid sequence, this sequence being able, moreover, to be modified by groups of a non-protein nature.
  • polypeptides may be grouped together in the expression "polypeptides”.
  • a particular nucleotide sequence is characterized in that it hybridizes with the four probes 16A, 18A, 66A and 66B, under stringent conditions.
  • the stringent conditions which are discussed here, are described in detail in the experimental part of the present application.
  • a particular sequence is characterized in that it hybridizes with the four probes 16A, 18A, 66A and 66B, under non-stringent conditions (less stringent compared to the conditions given below in title of stringent conditions).
  • the invention relates in particular to the nucleotide sequence characterized in that it is the 7 kb Xbal fragment of the plasmid pCMBl deposited at the CNCM under the number 1-1317.
  • the invention relates to a nucleotide sequence chosen from the sequences designated by the expressions Seql, Seq2.1, Seq2.2 or Seq3, represented in FIG. 6.
  • the invention also relates to a nucleotide sequence characterized in that it hybridizes with one of the sequences previously described, in that it is present in the DNA of a bacterium of the species Clostridium bifermentans, and in that '' It encodes a protein, polypeptide or peptide with the ability to participate in toxic activity against Diptera larvae and in particular against Mosquito or Blackfly larvae.
  • nucleotide sequences of the invention can be isolated for example from anaerobic bacteria of the Clostridium bifermentans species and in particular from the Clostridium bifermentans malaysia strain. They can also be synthesized chemically according to conventional techniques.
  • nucleotide sequences can be constituted by single or double stranded DNA, by cDNA or even by RNA.
  • the subject of the invention is also, according to a particular embodiment, a nucleotide sequence characterized in that it contains fragments coding for the following amino acid sequences: MNTNIFSTNL and / or for NNDEIYGEPDSSNI and / or for MNN (X) CEVNCE (X) T and / or for NASLGK and / or for FEL and / or for QVK and / or for ENTASGTE and / or for IEYHNNLR and / or for
  • the identification of amino acid sequences is carried out using the letter code for the designation of amino acids.
  • the invention also relates to a nucleotide fragment contained in one of the sequences defined above, characterized by the following properties:
  • a preferred nucleotide fragment coding for a 66 kDa protein is characterized in that it contains a sequence coding for the amino acid chain MNTNIFSTNL at its NH 2 -terminal end, and a sequence coding for the chain of amino acids NNDEIYGEPDSSNI as internal fragment.
  • nucleotide fragment according to the invention contained in one of the previously defined sequences, is characterized by the following properties:
  • Pi6 protein having a molecular weight of approximately 16 kDa.
  • a preferred fragment coding for a protein having a molecular weight of approximately 16 kDa is further characterized in that it contains a sequence coding for the chain of amino acids AY (R) QVKFHIEAVNEGLKI at its NH end. 2 -terminale and a sequence coding for the chain of amino acids DIPISPEDISK as an internal fragment.
  • nucleotide fragment according to the invention contained in one of the preceding sequences is characterized in that it codes for a P20 protein having a molecular weight of approximately 20 kDa, precursor of the P16 protein, P20 being synthesized during the sporulation phase of bacteria of the species C. bifermentans, in particular of Cbm.
  • the subject of the invention is also a fragment of nucleotides contained in one of the preceding sequences, characterized by the following properties:
  • Such a fragment can preferably be characterized in that it codes for a protein having a molecular weight of approximately 18 kDa and in that it contains a sequence coding for 1 • amino acid chain MNN (X) CEVNCE (X) has its NH 2 -terminal end and sequences coding for the amino acid sequences of NASL T-.WGK, FEL, QVK, ENTASGTE, and IEYHNNLR as internal fragments.
  • vectors characterized in that they contain a nucleotide sequence or a nucleotide fragment corresponding to the preceding definitions, this sequence or this fragment being inserted at a site which is not essential for the replication of the vector.
  • these vectors are plasmids.
  • a particular vector is characterized in that it is the plasmid pCBM1 deposited at the CNCM under the number "1-1317.
  • Another recombinant vector according to the invention is the plasmid pHT316 resulting from the introduction into the plasmid pHT315 (Arantes and Lereclus, 1991, Gene, 108: 115-119), of a 0.5 kb BamHI-EcoRI fragment (Nicolas et al (1993) FEMS Microbiol. Letter 106, 275-280) containing the promoter of the cytolysin of B_ ⁇ thurinqiensis (ard and Ellar, 1986, J. Mol. Biol., 191: 1-11).
  • This vector can be modified by the sequence Cbm.
  • the plasmid pHT316 advantageously allows the overproduction in Bt, of Cbm proteins, toxic towards insects.
  • a subject of the invention is also recombinant prokaryotic or eukaryotic host cells, characterized in that they contain a sequence or a fragment corresponding to one of the definitions given above, or else a vector of the invention under conditions allowing the cloning and / or expression of said sequence or of said fragment.
  • a particular host can be a bacterial cell, for example a strain of Clostridium bifermentans, a strain of Bacillus thurinqiensis or a strain of Bacillus sphaericus.
  • Another cell according to the invention can be a eukaryotic cell, for example a plant cell.
  • the subject of the invention is also a polypeptide or a composition of polypeptides, characterized in that it is encoded by a nucleotide sequence or a fragment of nucleotides defined above.
  • a particular polypeptide according to the invention is involved in a larvicidal activity with regard to Diptera larvae, in particular of Mosquitoes or Simulia, and is characterized by the following properties: - it is characteristic of an anaerobic bacterium of the species Clostridium bifermentans;
  • polypeptides of the invention comprise in particular a first protein characterized in that it has a molecular weight of approximately 16 kDa and in that it is the product of the expression in a recombinant cell of a fragment of nucleotide hybridizing with oligonucleotide 16A under stringent conditions, this fragment being contained in the Nsil-Xbal sequence of the Xbal fragment contained in the plasmid pCMB1 deposited at the CNCM under the number 1-1317.
  • Another protein of the invention is characterized in that it has a molecular weight of approximately 18 kDa and in that it is the product of the expression in a recombinant cell of a fragment of - nucleotides hybridizing with Oligonucleotide 18A under stringent conditions, this fragment being contained in the EcoRI-Xbal sequence of the Xbal fragment contained in the plasmid pCMB1 deposited at the CNCM under the number 1-1317 and described in FIG. 4A.
  • a third protein according to the invention is characterized in that it has a molecular weight of approximately 66 kDa and in that it is the product of the expression in a recombinant cell of a nucleotide fragment hybridizing with 1 ' oligonucleotide 66A and / or 66B under stringent conditions, this fragment being contained in the Xbal-EcoRI sequence of the Xbal fragment contained in the plasmid pCMB1 deposited at the CNCM under the number 1-1317.
  • the invention also relates to polypeptide fragments of the proteins P16, P18 or P66, or any fragment as obtained from the proteins previously defined when it is involved in larvicidal activity against Diptera larvae, especially mosquitoes or black flies.
  • Another sequence of interest is the amino acid sequence corresponding to the cbmll gene as shown in Figure 6-
  • a polypeptide of the invention is characterized in that it is recognized by antibodies directed against the P16 protein and / or by antibodies directed against the P18 protein and / or by antibodies to the P66 protein.
  • polypeptides of the invention are for example polypeptides comprising an amino acid sequence encoded by one of the sequences Seql, Seq2.1, Seq2.2 or Seq3 and recognized respectively by anti-P66 protein antibodies for the polypeptide comprising at least one of the Seql, Seq2.1 or seq2.2 sequences and with anti-P16 protein or anti-P18 protein antibodies for the polypeptide comprising the Seq3 sequence.
  • the application also relates to a polypeptide characterized in that it is modified by addition, deletion, substitution of amino acids since it retains the capacity of the corresponding unmodified polypeptide to intervene in the toxic activity with respect to Diptera larvae, in particular mosquitoes or black flies.
  • the present application also relates to polypeptide compositions characterized in that they comprise for example the P16 protein and the P18 protein or in that they comprise the P16, P18 and P66 proteins.
  • proteins are preferably in purified form, if necessary co-purified, either after isolation from a strain, either after expression in a recombinant cell host.
  • the invention also relates to a protein extract having a larvicidal activity vis-à-vis the larvae of Diptera, in particular of mosquitoes or black flies as obtained by:
  • composition of polypeptides according to the invention can also be characterized in that it has the larvicidal activity of a crude extract as defined above.
  • the present application also relates to monoclonal antibodies directed against a protein according to the definitions given above.
  • It also relates to a polyclonal antiserum characterized in that it is directed against a protein of the invention or against a composition of these proteins or against an extract as described above.
  • nucleotide sequences or fragments thereof according to 1 • invention also allow the preparation of nucleotide probes obtained by classification according to the conventional techniques fragments or sequences described above.
  • compositions with larvicidal activity comprising, as active principle, one or more polypeptides according to any one of the definitions given above.
  • Other compositions with larvicidal activity according to the invention can also be characterized in that they comprise, as active principle, recombinant cells corresponding to the definitions given above.
  • compositions can also comprise recombinant cells modified by sequences coding for one or more polypeptides with larvicidal activity of B. thurinqiensis and / or of B. sphaericus.
  • Figures 1 to 3 Immunological relationships, distribution and kinetics of synthesis of P66, P18 and P16.
  • Figure 1 SDS-PAGE of the toxic Cbm extract. The molecular weights of standard proteins are indicated in the right margin (in kDa).
  • Figure 2 Detection of proteins P66, P18 and P16 in Cbm and in non-toxic strains of C. bifermentans. 100 ⁇ l samples of spore-forming cultures were subjected to polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a nitrocellulose membrane and subjected to immunodetection with affinity purified IgG directed against P66 (A66), P18 ( A18) or P16 (A16). Well a, ⁇ Jb; well b, strain ATCC 638; well c, strain 744-83; well d, strain VPI 4407; well e, strain VPI 4413A.
  • Figure 3 Synthetic kinetics of P66, P18 and P16 during the sporulation of Cbm, at 34 "C (high) or at 42 ° C
  • P66 A66
  • P18 A18
  • P16 A16
  • Culture time in hours a, 4.5; b, 6 (corresponding to the sporulation t 0 ); c, 7.5; d, 9; e, 10.5; f, 13; g, 30.
  • the molecular weights of standard proteins are indicated in the margin (in kDa).
  • Figures 4A and 4B Structure of the plasmid pCBM1 and restriction map of the Xbal fragment.
  • Fragment Xbal Bg: BqlII; N: Nsil; P: PvuII; A:
  • H HindIII
  • S SphI
  • Ps PstI
  • Sal -.Sali
  • Bm
  • Xbal are fragments of the vector plasmid pHT304
  • the fragments on which the oligonucleotide probes hybridize are shown in hatched lines, under the Xbal fragment.
  • the arrow corresponds to the 5 ′ end of the P16 gene hybridizing with the 16A probe.
  • Figure 5 Location of the oligonucleotides used for the sequencing of the sequences read.
  • 16A ' complementary 16 A:: ace cat tgt cta tat gc
  • 16B ' complementary 16 B' * : ceg ata tet cet gaa ga 18 B * : oligo 18 B not degenerate: tct gta ccg gaa gca gt
  • 18 B ' complementary 18 b * : gct tcc ggt aca gaa gg
  • Figure 6 Sequences of nucleotides read.
  • 6C and 6D Seq.2.2, read with the primers 66B, 66D-A and
  • 6H and 6J SEq4, read with the universal primer R-40.
  • Figure 7 Amino acid sequence corresponding to the Cbmll gene and localization of the NH 2 -terminal and internal sequences of the proteins P18 and P16.
  • Figure 8 Location of copies of cbm 11 on the Xbal fragment.
  • Figure 9A and 9B Location of the Xbal fragment on the resident plasmid.
  • Line 1 CCC size marker from 16 to 2.06 Kb
  • Line 1 E. coli + pHT 304 (shuttle vector used)
  • Line 2 E. coli + pCBM 1 Probe used: total anti Cbm antibody.
  • Figures 11A, 11B and 11C Sequence homologies found between the protein Cbm 11 and the proteins described in the Swissprot database.
  • An extract was prepared from a Cbm culture carried out at 34 ° C. anaerobically in TYG medium (based on Biotrypcase 3%, yeast extract 2%, glucose 0.5 to 1%, cysteine hydrochloride 0, 05%) in a fermenter with a capacity of 6 liters, in the presence of a gas stream containing 5% H 2 , 5% C0 2 and 90% N 2 .
  • the sporulating bacterial culture was harvested after 16 h, at the end of the sporulation phase. The culture was then washed with 1M NaCl, rinsed twice with 20 mM Tris HCl, 5 mM EDTA, pH8 (TE buffer) and -reserved at -70 ° C until use.
  • the frozen pellets were thawed resuspended in a TE buffer treated in a sonicator for a total duration of l minute including a real time of sonication of 15 seconds on the ice (Branson sonicator, large probe, 40% output scale, duration of the cycle sonication ("duty cycle": 25%) and centrifuged at 5000 g for 15 min. The resulting supernatant (corresponding to the crude protein extract) was collected.
  • the protein concentration of the extract was determined using the Biorad protein test with bovine serum albumin as standard. Electrophoresis on polyacrylamide gel was carried out according to the technique of Laemmli, UK (1970) (Nature 227, 680-685) on 13% polyacrylamide gels.
  • the molecular weight markers used in this Analysis of proteins by SDS-PAGE under denaturing conditions are those of the Pharmacia protein electrophoresis kit (LMW Ref. 17-0446-01), containing the following proteins: phosphorylase B (of molecular mass 94,000 daltons), albumin (67,000) , ovalbumin (43,000), anhydrase (30,000) tryptic inhibitor (20,100) and alpha lactalbumin (14,000).
  • Polyclonal antisera against the crude Cbm protein extract were obtained in rabbits after two series of 10 to 15 intradermal microinjections of 500 ⁇ g of proteins emulsified in the complete Freund's adjuvant, 3 weeks apart. The rabbits received subcutaneous injections of a booster dose without adjuvant from Freund, 3 weeks later.
  • the IgGs were purified from the sera by precipitation with ammonium sulphate and chromatography on a DEAE-52 column (What ann) and then stored at 4 ° C.
  • Polyclonal antisera against the individualized denatured polypeptides P66, P18 and P16 were produced in rabbits as follows: the three proteins were separated by SDS-PAGE preparative electrophoresis and detected with 1M KC1. The strips were cut on the gels and the cut acrylamide strips were rinsed with 1 • deionized water, then immersed in water, emulsified with complete Freund's adjuvant and injected into the rabbits according to the technique described above. . After recovery of the antisera, the IgGs were purified by affinity on nitrocellulose bands containing the polypeptide used for injection into rabbits, according to the technique of Burke et al (1982). EMBO J. 1, 1621-1628. Enzymatic hydrolysis of Cbm extract
  • the larvicidal activity was tested on larvae of Anopheles stephensi at the third larval stage.
  • the capacity of the different antisera to inhibit the larvicidal activity of the extract was tested according to the following method: serial dilutions of the extract were incubated with a fixed volume of anti-extract IgG in a TE buffer. 20 ⁇ C for 1 hour. The toxicity of the untreated samples and control samples was tested using larvae of A. stephensi. Neutralization tests have been also carried out under the same conditions with the antisera or the antibodies purified by affinity, directed against the denatured proteins P66, P18 or P16. The proteins were immunoprecipitated from the extract with the antisera directed against the extract or against the individual proteins P66, P18 or P16 denatured by the technique of Howe et al, ((1982). Mol. Gen. Genet. 186 , 525-530), using Protein A Sepharose (Sigma) beads as carriers.
  • P66, P18 and P16 were detected with affinity purified IgG directed against each protein (A66, A18 and A16) and visualized with a detection system in Western blot ECL ® (Amersham).
  • the cultures were carried out at 34 ° C. and 42 ° C. in flasks in which a gas exchange was possible.
  • Samples of bacterial culture or protein extracts were tested on 20 larvae of Culex pipiens in the fourth larval stage and / or of A. stephensi in the third larval stage in plastic petri dishes with a capacity of 6 ml. Mortalities were recorded after 24 h and 48 h of exposure.
  • a crude extract of Cbm was tested with antisera directed against the crystals of B. thurinqiensis serovar israelensis 1884, B. thurinqiensis serovar aizawai 7.29, B. thurinqiensis serovar thurinqiensis 1715 and B. thurinqiensis serovar entomocidus HD9, as well as antisera HD9 crystal proteins of 42 and 51 kDa from B. sphaericus 2362.
  • the Cbm extract contained three major proteins with apparent molecular weights 66, 18 and 16 kDa, designated by the abbreviations P66, P18 and P16, as well as various minor components of a protein nature (Figs. 1 to 3). 1. Characterization of the proteins P66, P18 and P16
  • the extract obtained is toxic to the larvae of the Culex pipiens mosquitoes, Anopheles stephensi and Aedes aegypti.
  • P66, P18 and P16 are the main components of the toxic extracts of Cbm.
  • P66, P18 or P16 are not immunologically related ( Figure 2, lines a). P18 and P16 were only present in Cbm whereas a 66 kDa protein immunologically related to P66 from Cbm was detected in 4 non-entomopathogenic C. bifermentans strains tested ( Figure 2, lines b to e).
  • P18 and P16 in a culture carried out at 34 ° C. was concomitant with the sporulation of Cbm (FIG. 3) and the appearance of larvicidal activity.
  • P16 was synthesized in the form of a 20 kDa precursor (P20), gradually converted into a 16 kDa polypeptide during cell lysis (Fig. 3).
  • P18 and P20 / P16 are not detected immunologically in strains of C. bifermentans lacking larvicidal activity (Fig 2).
  • P18 and P20 / P16 are very weakly detected in Cbm cultured at 42 ° C, under conditions where the bacteria is not toxic (Fig 3).
  • P66 was detected in Cbm cells during the vegetative stage and during sporulation. In sporulating cells, other polypeptides immunologically related to P66 were detected, ranging from 25 to 66 kDa ( Figure 3); these polypeptides could be degradation products of P66.
  • IgG directed against denatured P66, P18 and / or P16 proteins did not neutralize the toxicity; on the other hand, antibodies against the crude fraction of the extract neutralized the toxicity. It is conceivable that IgG directed against denatured proteins were able to recognize the P66-P18-P16 complex, but did not recognize epitopes or domains involved in larvicidal activity.
  • Partial amino acid sequences of P66, P18 and PI6 were determined by microsequencing.
  • Amino acid sequences of proteins P66, P18 and PI6 These sequences were obtained by microsequencing, according to techniques commonly practiced by microsequencing laboratories. For each protein, i) the NH 2 -terminal sequences were produced and ii) one or more sequences of internal fragments obtained by tryptic hydrolysis of the protein. The sequences were determined with an Applied Biosystems 470 sequencer, from the proteins separated on SDS-PAGE and transferred to the membrane of PVDF Immobilon (Millipore).
  • the oligonucleotide probes were made from the underlined sequences
  • MNTNIFSTN 66A ATG AAT ACI AAT ATI
  • TTT TCI ACI AA (26mer) P66, internal DEWIYGEPD 66B * TC IGG TTC ICC ATA IAT CCA TTC ATC (26mer)
  • oligonucleotide probes were synthesized and used to screen a total DNA library of Cbm hydrolyzed by the enzyme Xbal, constructed in the shuttle plasmid pHT304 (Arantès & Lereclus, Gene. 1991. 108: 115-119 ).
  • a DNA library was constructed in a shuttle vector pHT304, constructed by O. Arantès and D. Lereclus, from a resident plasmid of B. thurinqiensis pHT 1030 and pUC19, capable of replicating in E. coli and in gram positive bacteria.
  • the total DNA of Cbm was hydrolyzed by Xbal, transferred to Hybond N + membrane (Amersham) after electrophoresis on agarose gel, and hybridized with each of the synthesized probes, labeled at the 3 ′ end with fluorescein ("ECL kit TM 3 * oligolabeling and detection Systems ", Amersham) according to the protocol recommended by the supplier under the following hybridization and stringency conditions:
  • Hybridization temperature 42 ° C, 15h, in a HYBAID hybridization oven (Cera-Labo) with 5-10 ng of probes / ml. Washes after hybridization: 2 times 5 minutes at room temperature in 5 x SSC, 0.1% SDS, then 2 times 15 minutes in 1 x SSC, 0.1% SDS.
  • the genomic DNA library of Cbm constructed in E. coli strain TG1 was screened by hybridization in colonies with the oligonucleotide 16A labeled at the 3 * end with fluorescein (ECL system 3 'oligolabeling Amersham). The screening made it possible to isolate a recombinant plasmid pCBM1 hybridizing with the oligonucleotide 16A but also with the oligonucleotides 66A, 66B and 18A.
  • a single 7 kb band was detected on the DNA hydrolyzed by Xbal by the probe corresponding to the NH2 terminal end of P16 (probe 16A). A weak reaction was also detected on this fragment with probes 18A and 66A.
  • the library was therefore constructed in E. coli TG1 with Xbal DNA fragments of around 7.5 kb size eluted on Prep-A-Gene (Biorad). Screening of the library (400 clones) by hybridization of the colonies on a Hybond N + filter with oligonucleotide 16A revealed 10 positive clones (same stringency conditions as described above). Analysis of the 10 recombinant plasmids by several restriction enzymes showed that they were all similar. One of the plasmids (pCBMl, 13.5 kb) was selected.
  • the plasmid pCBM1 has a size of 13.5 kb and therefore results from the insertion of an Xbal fragment of Cbm DNA of 7 kb at the Xbal cloning site of pHT304 (6.5 kb).
  • the regions with which the probes mentioned above hybridize were determined by Southern blot on the plasmid pCBM1 hydrolyzed by different restriction enzymes for the 4 probes.
  • the PCR technique using as primers the universal primer and the oligonucleotide 16A made it possible to locate the hybridization region from 16A to 2 kb from the 3 'end of the Xbal fragment (FIG. 4A).
  • the PCR was carried out using Taq polymerase and as primers, the oligonucleotide 16A and the universal primer (M13 universal sequehcing primer Pharmacia) and as template the plasmid pCBMl, with the following cycle:
  • the sequencing strategy used was that of "walking on the fragment".
  • the sequence was carried out according to the Sanger technique (Sanger, et al., 1977, Proc. Natl. Acad. Sci. USA, 74: 5463-5467) on the plasmid pCBM1 made single strand by denaturation with sodium hydroxide.
  • the extension was carried out using universal primers, reverse primers, or oligonucleotides corresponding to the sequences deduced from the amino acid sequences of the proteins P66, P18 and P16 or the nucleotide sequences read. All of the oligonucleotides used and their location on the DNA fragment are represented in FIG. 5.
  • the determination of the restriction sites contained in the sequence of the gene coding for the 66 kDa protein made it possible to precisely locate the position of the start of the 66kDa gene on the Xbal fragment; approximately 95% of the gene is present on the cloned fragment.
  • the 16 kDa protein would in fact be a protein with a calculated molecular mass of 8.9 kDa; it could result from the 11 kDa protein by cleavage of the 17 amino acids NH 2 -terminals.
  • Hybridization experiments were carried out in parallel on the plasmid DNA and the total DNA of Cbm using as probe the Xbal fragment of 7 kb; these experiments were carried out using the technique "Direct nucleic acid labeling system" ECL (Amersham) of cold probes, according to the indications of the supplier. The results obtained made it possible to locate this fragment on a resident plasmid of approximately 13 kb (FIGS. 9A and 9B).
  • the study of the expression of the cloned genes in the plasmid pCBM1 was carried out in E. coli by immunodetection using cold probes.
  • the extracts of E. coli were prepared in the following manner: the clones TG1 (pHT304) and TG1 (pCBMl) were cultured in LB medium. This experience brought evidence the synthesis in this recombinant of a unique 20 kDa protein reacting specifically with the anti-protein 18 kDa antibody ( Figure 10). No product corresponding to the expression of the 66 kDa gene could be viewed.

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  • Gastroenterology & Hepatology (AREA)
  • Biotechnology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
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EP94920513A 1993-06-25 1994-06-24 Fragment d'adn de clostridium bifermentans portant les genes codant pour des proteines associees a une activite insecticide Withdrawn EP0705337A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
FR9307795 1993-06-25
FR9307795A FR2706908B1 (enrdf_load_stackoverflow) 1993-06-25 1993-06-25
PCT/FR1994/000768 WO1995000639A1 (fr) 1993-06-25 1994-06-24 Fragment d'adn de clostridium bifermentans portant les genes codant pour des proteines associees a une activite insecticide

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EP0705337A1 true EP0705337A1 (fr) 1996-04-10

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US (1) US5830722A (enrdf_load_stackoverflow)
EP (1) EP0705337A1 (enrdf_load_stackoverflow)
JP (1) JPH08511686A (enrdf_load_stackoverflow)
CA (1) CA2166124A1 (enrdf_load_stackoverflow)
FR (1) FR2706908B1 (enrdf_load_stackoverflow)
WO (1) WO1995000639A1 (enrdf_load_stackoverflow)

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US20030044979A1 (en) * 2001-04-05 2003-03-06 Isis Pharmaceuticals Inc. Antisense modulation of phospholipid scramblase I expression
US6251604B1 (en) 1999-08-13 2001-06-26 Genopsys, Inc. Random mutagenesis and amplification of nucleic acid

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IL86623A0 (en) * 1988-06-05 1988-11-30 Biotechnological Applic Ltd Bacilli and insecticidal compositions containing the same
CA2059898C (en) * 1991-02-21 2004-10-19 Gregory A. Bradfisch Biologically active bacillus thuringiensis isolates and gene encoding a coleopteran-active toxin

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Title
See references of WO9500639A1 *

Also Published As

Publication number Publication date
JPH08511686A (ja) 1996-12-10
FR2706908A1 (enrdf_load_stackoverflow) 1994-12-30
CA2166124A1 (fr) 1995-01-05
WO1995000639A1 (fr) 1995-01-05
US5830722A (en) 1998-11-03
FR2706908B1 (enrdf_load_stackoverflow) 1995-09-01

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