# Patent

## Title
Nucleotide sequences coding for polypeptides endowed with a larvicidal activity towards Lepidoptera

## Abstract

This invention relates to vectors, bacterial strains, and methods for the cloning and expression of a polypeptide having larvicidal activity. In particular, the invention relates to vectors, bacterial strains and methods for the cloning and expression of the N-terminal region of a polypeptide toxic against the larvae of Lepidoptera of the Noctuidae family, preferably against S. littoralis.

## Background
This application is a division of application Ser. No. 09/037,621, filed Mar. 10, 1998, issued as U.S. Pat. No. 6,310,035, on Oct. 30, 2001, which is a divisional of Ser. No. 08/461,551 filed Jun. 5, 1995, issued as U.S. Pat. No. 5,792,928, on Aug. 11, 1998, which is a divisional of Ser. No. 05/251,652 filed May 31, 1994, now abondoned, which is a continuation of Ser. No. 08/094,382 filed Jul. 21, 1993, now abandoned, which is a continuation of Ser. No. 07/458,754 filed Dec. 11, 1989, now abandoned, which claim priority, to EPO 87 08090, filed Jun. 10, 1987 and French application 88 401121.4, filed May 6, 1988, which are incorporated herein by reference.


The subject of the invention is nucleotide sequences coding for polypeptides endowed with a larvicidal activity towards Lepidoptera.
It relates more particularly to agents, in particular nucleotide sequences, polypeptides or even vectors, or bacterial strains modified by these sequences and expressing polypeptides making it possible to prepare larvicidal compositions active against Lepidoptera, preferably against Spodoptera littoralis (hereafter S.littoralis) or Mamestra brassicae (hereafter designated by M.brassicae) or capable of transforming the plants to be treated in conferring on them this type of activity.
It is known that most of the isolates of B.thuringiensis show a toxic activity with regard to larvae of more than a hundred species of Lepidoptera.
This activity results from the capacity of the strains of B.thuringiensis to synthesize, at the moment of sporulation, crystalline inclusions of protein nature, or δ-endotoxins, under the control of one or several types of gene.
It has been shown that the activity of these polypeptides is contained in the NH2-terminal half or N-terminus of the protein.
The studies carried out have shown the high specificity of the δ-endotoxins towards larvae of a given species.
On account of this high specificity, many species of Lepidoptera, in particular of the family of the Noctuidae, react only weakly to commercial preparations of available B.thuringiensis. 
It is so in particular for the species S.littoralis, a poly-phagous insect which constitutes the principal parasite of cotton and other industrially important crops. Among these crops, mention should be made of maize, the castor oil plant, tobacco, the groundnut, fodder plants, such as clover or alfalfa, or also market garden produce such as the cabbage or the tomato.
Hence, one can imagine the interest of disposing of agents targeting specifically and effectively the family of the Noctuidae and in particular S.littoralis or M.brassicae. 
The genes for δ-endotoxins hitherto identified do not code for a polypeptide preferentially active with regard to S.littoralis. 
The search by the inventors for a sequence of nucleotides coding for a polypeptide preferably active against the Noctuidae, more especially against S.littoralis, has led them to study the natural isolates of two strains of B.thuringiensis, the larvicidal activity of which on S.littoralis appears to be higher than that of the industrial preparations made starting from other strains of B.thuringiensis. 
The species in question are aizawai 7-29 and entomocidus 6-01.
The study of these isolates has made it possible to demonstrate the existence of several genes for δ-endotoxins of different structures and different specificities, of which two genes preferentially active against P.brassicae but not very active against the Noctuida of cotton and a gene inactive against P.brassicae and S.littoralis. 
By studying the total DNA of these isolates and by carrying out appropriate hybridizations, followed by the cloning of the fragments identified by hybridization, the inventors have observed that it is possible to isolate nucleotide sequences implicated in genes for δ-endotoxins coding for polypeptides active, preferably, against S.littoralis. 
Thus, the aim of the invention is to provide nucleotide sequences capable of coding for at least the NH2-terminal part of a δ-endotoxin toxic against the Noctuidae and preferably against S.littoralis or M.brassicae. 
It also has the aim of providing a polypeptide toxic with regard to the Noctuidae.
Furthermore, the invention relates to a procedure for obtaining such a sequence and a polypeptide showing the desired activity as well as the intermediate agents such as vectors and bacterial strains which can be utilized for obtaining the polypeptide.
In addition, the invention relates to the uses of these sequences and polypeptides for the development of larvicidal compositions with regard to the Noctuidae, in particular S.littoralis and for the transformation of the plants likely to be infected by these larvae.
The invention relates to a sequence of nucleotides coding for at least a part of the N-terminal region of a polypeptide toxic specifically against the larvae of Lepidoptera of the Noctuidae family, and preferably against S.littoralis, characterized by its capacity of hybridization with a gene capable of expressing a polypeptide toxic towards larvae of S.littoralis. 
According to another aspect of the invention, the nucleotide sequence is characterized in that it is carried by a sequence of nucleotides of about 3 kb such as obtained by in vitro genetic recombination of sequences of nucleotides of B.thuringiensis capable of hybridizing with probes 1, 2 and 3 of pHTA2 shown in FIG. 2. The fragment of 3 kb corresponds more particularly to the restriction fragment HindIII-PstI.
The sequences of nucleotides of the invention are, in addition, characterized in that they contain sites in the following order: HindIII-HincII-BglII-KpnI-HindIII-PstI.
In a preferred manner, these sequences of nucleotides are obtained by in vitro genetic recombination of DNA sequences derived from at least one strain of B.thuringiensis. In a variant of the embodiment of the invention, two different strains of B.thuringiensis are utilized.
Strains of B.thuringiensis particularly suited for obtaining these sequences of nucleotides are the strains corresponding to aizawai 7-29 and entomocidus 6-01, deposited on Apr. 21, 1987 under the No. I-661 and No. I-660, respectively, with the National Collection of Cultures of Microorganisms (N.C.C.M.) in Paris.
In an advantageous manner, the sequences of nucleotides of the invention code for a polypeptide capable of forming an immunological complex with antibodies directed against polypeptides showing the larvicidal activity with regard to S.littoralis.
A sequence of nucleotides according to the invention is characterized in that it has the capacity to hybridize with a probe formed from the sequence (I) showing the following chain arrangement (nucleotides 52-990 of SEQ ID NO:1):








    52



GTC TAC TTG ACA CCG GTA CGA ACA TAA TCT GTC AAT


                                    112


TTT AAA TAT GGG GCA TAT ATT GAT ATT TTA TAA AAT


TTG TTA CGT TTT TTG TAT TTT TTC ATA AGA TCT GTC


                    172


ATA TGT ATT AAA TCG TGG TAA TGA AAA ACA GTA TCA


AAC TAT CAG AAC TTT GGT AGT TTA ATA AAA AAA CGG


    232


AGG TAT TTT ATG GAG GAA AAT AAT CAA AAT CAA TGC


                                    292


ATA CCT TAC AAT TGT TTA AGT AAT CCT GAA GAA GTA


CTT TTG GAT GGA GAA CGG ATA TCA ACT GGT AAT TCA


                    352


TCA ATT GAT ATT TCT CTG TCA CTT GTT CAG TTT CTG


GTA TCT AAC TTT GTA CCA GGG GGA GGA TTT TTA GTT


    412


GCA TTA ATA GAT TTT GTA TGG GGA ATA GTT GGC CCT


                                    472


TCT CAA TGG GAT GCA TTT CTA GTA CAA ATT GAA CAA


TTA ATT AAT GAA AGA ATA GCT GAA TTT GCT AGG AAT


                    532


GCT GCT ATT GCT AAT TTA GAA GGA TTA GGA AAC AAT


TTC AAT ATA TAT GTG GAA GCA TTT AAA GAA TGG GAA


    592


GAA GAT CCT AAT AAT CCA GAA ACC AGG ACC AGA GTA


                                    652


ATT GAT CGC TTT CGT ATA CTT GAT GGG CTA CTT GAA


AGG GAC ATT CCT TCG TTT CGA ATT TCT GGA TTT GAA


                    712


GTA CCC CTT TTA TCC GTT TAT GCT CAA GCG GCC AAT


CTG CAT CTA GCT ATA TTA AGA GAT TCT GTA ATT TTT


    772


GGA GAA AGA TGG GGA TTG ACA ACG ATA AAT GTC AAT


                                    832


GAA AAC TAT AAT AGA CTA ATT AGG CAT ATT GAT GAA


TAT GCT GAT CAC TGT GCA AAT ACG TAT AAT CGG GGA


                    892


TTA AAT AAT TTA CCG AAA TCT ACG TAT CAA GAT TGG


ATA ACA TAT AAT CGA TTA CGG AGA GAC TTA ACA TTG


    952


ACT GTA TTA GAT ATC GCC GCT TTC TTT CCA AAC TAT


GAC






Sequences of nucleotides coding for at least a part of the N-terminal region of a polypeptide toxic specifically towards larvae of Lepidoptera of the Noctuidae family, and preferably towards S.littoralis, are characterized in that they contain the chain arrangement (I) defined above.
In an advantageous manner, the sequence of nucleotides characterized by the chain arrangement defined above codes for a part of a polypeptide having a higher larvicidal activity towards S.littoralis than that of the polypeptides encoded by natural isolates presently known for their effects against S.littoralis. 
The study of this sequence of nucleotides shows that it is characterized by an initiation codon ATG situated at position 241 starting from which an open reading frame of 750 nucleotides has been identified.
This sequence is also characterized by a GGAGG attachment site for ribosomes at positions 230 to 234.
According to another feature, the sequence of nucleotides of the invention is characterized in that it contains, upstream from the ATG codon, a sequence going from the nucleotide at position 137 to the nucleotide at position 177, strongly homologous with the region found by Wong et al. (1983) and described in (16) upstream from the gene for the crystal of the strain kurstaki HD1 Dipel (BTK) and for which the authors have shown that it contains three promoters BtI, BtII and Ec which are functional in B.thuringiensis and E.coli, respectively. The homology of these sequences is about 70%.
The invention also relates to a sequence of nucleotides coding for the following sequence (II) of amino acids (amino acids 1-250 of SEQ ID NO:2):








                            MET GLU GLU ASN ASN



GLN ASN GLN CYS ILE PRO TYR ASN CYS LEU SER ASN


PRO GLU GLU VAL LEU LEU ASP GLY GLU ARG ILE SER


THR GLY ASN SER SER ILE ASP ILE SER LEU SER LEU


VAL GLN PHE LEU VAL SER ASN PHE VAL PRO GLY GLY


PHE LEU VAL GLY LEU ILE ASP PHE VAL TRP GLY ILE


VAL GLY PRO SER GLN TRP ASP ALA PHE LEU VAL GLN


ILE GLU GLN LEU ILE ASN GLU ARG ILE ALA GLU PHE


ALA ARG ASN ALA ALA ILE ALA ASN LEU GLU GLY LEU


GLY ASN ASN PHE ASN ILE TYR VAL GLU ALA PHE LYS


GLU TRP GLU GLU ASP PRO ASN ASN PRO GLU THR ARG


THR ARG VAL ILE ASP PRO PHE ARG ILE LEU ASP GLY


LEU LEU GLU ARG ASP ILE PRO SER PHE ARG ILE SER


GLY PHE GLU VAL PRO LEU LEU SER VAL TYR ALA GLN


ALA ALA ASN LEU HIS LEU ALA ILE LEU ARG ASP SER


VAL ILE PHE GLY GLU ARG TRP GLY LEU THR THR ILE


ASN VAL ASN GLU ASN TYR ASN ARG LEU ILE ARG HIS


ILE ASP GLU TYR ALA ASP HIS CYS ALA ASN THR TYR


ASN ARG GLY LEU ASN ASN LEU PRO LYS SER THR TYR


GLN ASP TRP ILE THR TYR ASN ARG LEU ARG ARG ASP


LEU THR LEU THR VAL LEU ASP ILE ALA ALA PHE PHE


PRO ASN TYR ASP






A better identification of the sequence of nucleotides isolated from the above strains, deposited with the NCCM has made it possible to observe that the nucleotide situated at position 273 is guanine (G), the amino acid resulting from the GTA codon thus being valine.
Now, the reading of the nucleotide corresponding to this position 273 in the application FR.8708090 of Jun. 10, 1987 had led to reporting thymine (T) and leucine as amino acid resulting from the TTA codon.
Another sequence of nucleotides of the invention is characterized by its capacity of hybridization with a probe formed from the sequence (III) showing the following chain arrangement (SEQ ID NO:1):








    1



AAG CTT CAA TAG AAT CTC AAA TCT CGA TGA CTG CTT AGT CTT TTT AAT ACT GTC TAC TTG ACA GGG GTA GGA ACA TAA








TCG GTC AAT TTT









   91



AAA TAT GGG GCA TAT ATT GAT ATT TTA TAA AAT TTG TTA CGT TTT TTG TAT TTT TTC ATA AGA TGT GTC ATA TGT ATT








AAA TCG TGG TAA









  161



TGA AAA ACA GTA TCA AAC TAT CAG AAC TTT GGT AGT TTA ATA AAA AAA CGG AGG TAT TTT ATG GAG GAA AAT AAT CAA








AAT CAA TGC ATA









  271



CCT TAC AAT TGT TTA AGT AAT CCT GAA GAA GTA CTT TTG GAT GGA GAA CGG ATA TCA ACT GGT AAT TCA TCA ATT GAT








ATT TCT CTG TCA









  361



CTT GTT CAG TTT CTG GTA TCT AAC TTT GTA CCA GGG GGA GGA TTT TTA GTT GGA TTA ATA GAT TTT GTA TGG GGA ATA








GTT GGC CCT TCT









  431



CAA TGG GAT GCA TTT CTA GTA CAA ATT GAA CAA TTA ATT AAT GAA AGA ATA GCT GAA TTT GCT AGG AAT GCT GCT ATT








GCT AAT TTA GAA









  541



GGA TTA GGA AAC AAT TTC AAT ATA TAT GTG GAA GCA TTT AAA GAA TGG GAA GAA GAT CCT AAT AAT CCA GCA ACC AGG








ACC AGA GTA ATT









  631



GAT CGC TTT CGT ATA CTT GAT GGG CTA CTT GAA AGG GAC ATT CCT TCG TTT CGA ATT TCT GGA TTT GAA GTA CCC CTT








TTA TCC GTT TAT









  721



GCT CAA GCG GCC AAT CTG CAT CTA GCT ATA TTA AGA GAT TCT GTA ATT TTT GGA GAA AGA TGG GGA TTG ACA ACG ATA








AAT GTC AAT GAA









  691



AAC TAT AAT AGA CTA ATT AGG CAT ATT GAT GAA TAT GCT GAT CAC TGT GCA AAT ACG TAT AAT CGG GGA TTA AAT AAT








TTA CCG AAA TCT









  701



ACG TAT CAA GAT TGG ATA ACA TAT AAT CGA TTA CGG AGA GAC TTA ACA TTG ACT GTA TTA GAT ATC ACC GCT TTC TTT








CCA AAC TAT GAC









  991



AAT AGG AGA TAT CCA ATT CAG CCA GTT GGT CAA CTA ACA AGG GAA GTT TAT ACG GAC CCA TTA ATT AAT TTT AAT CCA








CAG TTA CAG TCT









 1081



GTA GCT CAA TTA CCT ACT TTT AAC GTT ATG GAG AGC AGC GCA ATT AGA AAT CCT CAT TTA TTT GAT ATA TTG AAT AAT








CTT ACA ATC TTT









 1171



ACG GAT TGG TTT AGT GTT GGA CGC AAT TTT TAT TGG GGA GGA CAT CGA GTA ATA TCT AGC CTT ATA GGA GGT GGT AAC








ATA ACA TCT CCT









 1261



ATA TAT GGA AGA GAG GCG AAC CAG GAG CCT CCA AGA TCC TTT ACT TTT AAT GGA CCG GTA TTT AGG ACT TTA TCA ATT








CCT ACT TTA CGA









 1381



TTA TTA CAG CAA CCT TGC CAG CGC CAC CAT TTT AAT TTA CGT GGT GGT GAA GGA GTA GAA TTT TCT ACA CCT ACA AAT








AGC TTT ACG TAT









 1447



CGA GGA AGA GGT ACG GTT GAT TCT TTA ACT GAA TTA CCG CCT GAG GAT AAT AGT GTG CCA CCT CGC GAA GGA TAT AGT








CAT CGT TTA TGT









 1571



CAT GCA ACT TTT GTT CAA AGA TCT GGA ACA CCT TTT TTA ACA ACT GGT GTA GTA TTT TCT TGG ACG CAT CGT AGT GCA








ACT CTT ACA AAT









 1621



ACA ATT GAT CCA GAG AGA ATT AAT CAA ATA CCT TTA GTG AAA GGA TTT AGA GTT TGG GGG GGC ACC TCT GTC ATT ACA








GGA CCA GGA TTT









 1711



ACA GGA GGG GAT ATC CTT CGA AGA AAT ACC TTT GGT GAT TTT GTA TCT CTA CAA GTC AAT ATT AAT TCA CCA ATT ACC








CAA AGA TAC CGT









 1801



TTA AGA TTT CGT TAC GCT TCC AGT AGG GAT GCA CGA ATT ATA GTA TTA ACA GGA GCG GCA TCC ACA GGA GTG GGA GGC








CAA GTT AGT GTA









 1891



GAT ATG CCT CTT CAG AAA ACT ATG GAA ATA GGG GAG AAC TTA ACA TCT AGA ACA TTT AGA TAT ACC GAT TTT AGT AAT








CCT TTT TCA TTT









 2981



AGA GCT AAT CCA GAT ATA ATT GGG ATA AGT GAA CAA CCT CTA TTT GGT GCA GGT TCT ATT AGT AGC GGT GAA CTT TAT








ATA GAT AAA ATT









 2071



GAC ATT ACT CTA GCA GAT GCA ACA TTT GAA GCA GAA TCT GAT TTA GAA AGA GCA CAA AAG GCG GTG AAT GCC CTG TTT








ACT TCT TCC AAT









 2161



CAA ATC GGG TTA AAA ACC GAT GTG ACG GAT TAT CAT ATT GAT CAA GTA TCC AAT TTA GTG GAT TGT TTA TCA GAT GAA








TTT TGT CTG GAT









 2251



GCA AAG CGA GAA TTG TCC GAG AAA GTC AAA CAT GCG AAG CGA CTC AGT GAT GAG CGG AAT TTA CTT CAA GAT CCA AAC








TTC AGA GGG ATC









 2341



AAT AGA CAA CCA GAC CGT GGC TGG AGA GGA AGT ACA GAT ATT ACC ATC CAA GGA GGA GAT GAC GTA TTC AAA GAG AAT








TAC GTC ACA CTA









 2431



CCG GGT ACC GTT GAT GAG TGC TAT CCA ACG TAT TTA TAT CAG AAA ATA GAT GAG TCG AAA TTA AAA GCT TAT ACC CGT








TAT GAA TTA AGA









 2521



GGG TAT ATC GAA GAT AGT CAA GAC TTA GAA ATC TAT TTG ATC GCG TAC AAT GCA AAA CAC GAA ATA GTA AAT GTG CCA








GGC ACG GGT TCC









 2611



TTA TGG CCG CTT TCA GCC CAA AGT CCA ATC GGA AAG TGT GGA GAA CCG AAT CGA TGC GCG CCA CAC CTT GAA TGG AAT








CCT GAT CTA GAT









 2701



TGT TCC TGC AG






In a distinctive manner, sequences of nucleotides of the invention coding for a polypeptide toxic specifically towards larvae of Lepidoptera of the Noctuidae family, and preferably toward S.littoralis comprise or are constituted by the chain arrangement (III) previously defined.
The chain arrangement (III), comprised in the sequence of nucleotides of the invention contains 2711 nucleotides. This fragment includes in particular the potential promoter of the gene of the δ-endotoxin active on S.littoralis. 
Sequences of nucleotides modified in relation to the chain arrangements (I) or (III) described above naturally enter into the framework of the present invention to the extent to which these modifications do not generate appreciable variations of the toxicity of the polypeptide coded by the modified sequence towards S.littoralis. 
These modifications may, for example, consist of deletions, substitutions, recombinations.
Thus, the sequences of nucleotides (I) and (III) contain at their position 611 a variable nucleotide corresponding to adenine (A) in the sequence (I) and to cytosine (C) in the sequence (III). These nucleotides enter into the composition of the respective codons GAA and GCA which code respectively for the amino acids glutamic acid (GLU) and alanine (ALA) in the respective sequences II and IV.
Similarly, any sequence of nucleotides which can hybridize with that of the chain arrangements (I) or (III) such as obtained by reverse enzymatic transformation of the corresponding RNA or even by chemical synthesis also enter into the framework of the definitions of the invention.
The sequence of nucleotides of formula (III) starts with a ATG initiation codon situated at position 241 and which represents the start of an open reading frame of 2470 nucleotides.
The invention also relates to sequence of nucleotides characterized in that it codes for a polypeptide containing the sequence (IV) of amino acids below (SEQ ID NO:2):








                                                                                MET GLU GLU ASN ASN GLN









ASN GLN CYS ILE









    271



PRO TYR ASN CYS LEU SER ASN PRO GLU GLU VAL LEU LEU ASP GLY GLU ARG ILE SER THR GLY ASN SER SER ILE ASP








ILE SER LEU SER









    361



LEU VAL GLN PHE LEU VAL SER ASN PHE VAL PRO GLY GLY GLY PHE LEU VAL GLY LEU ILE ASP PHE VAL TRP GLY ILE








VAL GLY PRO SER









    451



GLN TRP ASP ALA PHE LEU VAL GLN ILE GLU GLN LEU ILE ASN GLU ARG ILE ALA GLU PHE ALA ARG ASN ALA ALA ILE








ALA ASN LEU GLU









    541



GLY LEU GLY ASN ASN PHE ASN ILE TYR VAL GLU ALA PHE LYS GLU TRP GLU GLU ASP PRO ASN ASN PRO ALA THR ARG








THR ARG VAL ILE









    631



ASP ARG PHE ARG ILE LEU ASP GLY LEU LEU GLU ARG ASP ILE PRO SER PHE ARG ILE SER GLY PHE GLU VAL PRO LEU








LEU SER VAL TYR









    721



ALA GLN ALA ALA ASN LEU HIS LEU ALA ILE LEU ARG ASP SER VAL ILE PHE GLY GLU ARG TRP GLY LEU THR THR ILE








ASN VAL ASN GLU









    811



ASN TYR ASN ARG LEU ILE ARG HIS ILE ASP GLU TYR ALA ASP HIS CYS ALA ASN THR TYR ASN ARG GLY LEU ASN ASN








LEU PRO LYS SER









    901



THR TYR GLN ASP TRP ILE THR TYR ASN ARG LEU ARG ARG ASP LEU THR LEU THR VAL LEU ASP ILE ALA ALA PHE PHE








PRO ASN TYR ASN









    981



ASN ARG ARG TYR PRO ILE GLN PRO VAL GLY GLN LEU THR ARG GLU VAL TYR THR ASP PRO LEU ILE ASN PHE ASN PRO








GLN LEU GLN SER









  1081



VAL ALA GLN LEU PRO THR PHE ASN VAL MET GLU SER SER ALA ILE ARG ASN PRO HIS LEU PHE ASP ILE LEU ASN ASN








LEU THR ILE PHE









  1171



THR ASP TRP PHE SER VAL GLY ARG ASN PHE TYR TAP GLY GLY HIS ARG VAL ILE SER SER LEU ILE GLY GLY GLY ASN








ILE THR SER PRO









  1261



ILE TYR GLY ARG GLU ALA ASN GLN GLU PRO PRO ARG SER PHE THR PHE ASN GLY PRO VAL PHE ARG THR LEU SER ILE








PRO THR LEU ARG









  1351



LEU LEU GLN GLN PRO CYS GLN ARG HIS HIS PHE ASN LEU ARG GLY GLY GLU GLY VAL GLU PHE SER THR PRO THR ASN








SER PHE THR TYR









  1441



ARG GLY ARG GLY THR VAL ASP SER LEU THR GLU LEU PRO PRO GLU ASP ASN SER VAL PRO PRO ARG GLU GLY TYR SER








HIS ARG LEU CYS









  1531



HIS ALA THR PHE VAL GLN ARG SER GLY THR PRO PHE LEU THR THR GLY VAL VAL PHE SER TRP THR HIS ARG SER ALA








THR LEU THR ASN









  1621



THR ILE ASP PRO GLU ARG ILE ASN GLN ILE PRO LEU VAL LYS GLY PHE ARG VAL TRP GLY GLY THR SER VAL ILE THR








GLY PRO GLY PHE









  1711



THR GLY GLY ASP ILE LEU ARG ARG ASN THR PHE GLY ASP PHE VAL SER LEU GLN VAL ASN ILE ASN SER PRO ILE THR








GLN ARG TYR ARG









  1601



LEU ARG PHE ARG TYR ALA SER SER ARG ASP ALA ARG VAL ILE VAL LEU THR GLY ALA ALA SER THR GLY VAL GLY GLY








GLN VAL SER VAL









  1891



ASN MET PRO LEU GLN LYS THR MET GLU ILE GLY GLU ASN LEU THR SER ARG THR PHE ARG TYR THR ASP PHE SER ASN








PRO PHE SER PHE









  1901



ARG ALA ASN PRO ASP ILE ILE GLY ILE SER GLU GLN PRO LEU PHE GLY ALA GLY SER ILE SER SER GLY GLU LEU TYR








ILE ASP LYS ILE









  2071



GLU ILE ILE LEU ALA ASP ALA THR PHE GLU ALA GLU SER ASP LEU GLU ARG ALA GLN LYS ALA VAL ASN ALA LEU PHE








THR SER SER ASN









  2161



GLN ILE GLY LEU LYS THR ASP VAL THR RSP TYR HIS ILE ASP GLN VAL SER ASN LEU VAL ASP CYS LEU SER ASP GLU








PHE CYS LEU ASP









  2251



GLU LYS ARG GLU LEU SER GLU LYS VAL LYS HIS ALA LYS ARG LEU SER ASP GLU ARG ASN LEU LEU GLN ASP PRO ASN








PHE ARG GLY ILE









  2341



ASN ARG GLN PRO ASP ARG GLY TRP ARG GLY SER THR ASP ILE THR ILE GLN GLY GLY ASP ASP VAL PHE LYS GLU ASN








TYR VAL THR LEU









  2431



PRO GLY THR VAL ASP GLU CYS TYR PRO THR TYR LEU TYR GLN LYS ILE ASP GLU SER LYS LEU LYS ALA TYR THR ARG








TYR GLU LEU ARG









  2521



GLY TYR ILE GLU ASP SER GLN ASP LEU GLU ILE TYR LEU ILE ALA TYR ASN ALA LYS HIS GLU ILE VAL ASN VAL PRO








GLY THR GLY SER









  2611



LEU TRP PRO LEU SER ALA GLN SER PRO ILE GLY LYS CYS GLY GLU PRO ASN ARG CYS ALA PRO HIS LEU GLU TRP ASN








PRO ASP LEU ASP









  2701



CYS SER CYS






The invention also relates to recombinant expression and cloning vectors comprising more particularly at least one sequence of nucleotides such as that defined above, in particular at least a part of the sequence of about 3 kb.
A specific recombinant vector is, for example, a plasmid containing the HindIII-PstI fragment of the sequence of nucleotides of the invention, inserted in a vector pUC9. A first preferred vector is the plasmid pHT71, the construction of which is reported in the assemblies below, which comprises a HindIII-PstI DNA fragment according to the invention constituted uniquely of DNA derived from the strain aizawai 7-29.
Another recombinant vector is constituted by the plasmid pHT 671, the construction of which is given in FIG. 4. This plasmid contains a chimeric HindIII-PstI fragment, obtained by fusing a HindIII-HindII fragment of 1.1 kb derived from the strain entomocidus 6-01 with a HincII-PstI fragment of 1.9 kb derived from the strain aizawai 7-29.
The modified bacterial strains which contain one of the nucleotide sequences defined above or also a recombinant expression vector and cloning previously defined, and preferably the plasmid pHT671 or the plasmid pHT71, also enter into the framework of the invention.
The invention also relates to a polypeptide toxic towards larvae of Lepidoptera and in a preferential manner towards S.littoralis, which attack cotton leaves or other crops such as those listed above, characterized in that it is capable of forming an immunological complex with antibodies directed against polypeptides with larvicidal activity towards S.littoralis. 
The invention relates more particularly to the NH2-terminal part of this polypeptide which contains the larvicidal activity.
The extremity of the active NH2-terminal part corresponds to the sequence (II) of amino acids given above.
A preferred polypeptide of the invention is that which corresponds to this sequence (II) and corresponds to the sequence (IV) of amino acids given in the preceding pages. This polypeptide corresponding to the sequence (IV) contains 823 amino acids. Its calculated molecular mass is 92906 Da.
This sequence of δ-endotoxin was compared with amino acid sequences of δ-endotoxins derived from other strains of B.thuringiensis active on the Lepidoptera and the genes of which have been isolated and sequenced previously: the δ-endotoxins in question are those of the strains kurstaki HD1 (19), kurstaki HD73 (20), berliner 1715 (21) and (22) Sotto (23) and aizawai IPL7 (24).
The results of these comparisons indicate that all are different in the second quarter of the molecule (amino acids 281 to 620) whereas the NH2-terminal part (amino acids 1 to 280) and the COOH-terminal domain (amino acids 621 to 1175) of the protein are highly conserved and differ only by several amino acids. On the other hand, the δ-endotoxin corresponding to the sequence (IV) above shows considerable differences from the other δ-endotoxins both in the NH2-terminal part (amino acids 1 to 280) and in the second quarter of the molecule (amino acids 281 to 620). The results of these comparisons indicate again that the NH2-terminal half of the molecule (amino acids 1 to 620) which corresponds to the toxic fraction of the protein only show 46% homology with the other δ-endotoxins. The most important differences are located in the second half of the toxic part of the molecule (amino acids 281 to 620) with only 36% of identical amino acids, the NH2-terminal part (amino acids 1 to 280) itself showing 58% of amino acids identical with the other δ-endotoxins. Such considerable differences have never been observed up to now in the NH2-terminal part of the toxic fraction of the molecule among the δ-endotoxins active on the Lepidoptera.
In order to obtain a sequence of nucleotides entering into the framework of the invention, coding for at least the active part of a polypeptide showing a specific toxicity towards larvae of Lepidoptera of the Noctuidae family, and preferably towards S.littoralis, recourse is had, in conformity with the invention, to the following steps, namely:

    
    
        the carrying out of a molecular hybridization between, on the one hand, a nucleotide sequence of a strain of B.thuringiensis active against S.littoralis and, on the other, at least two nucleotide sequences, used as probes, derived from the 5′ part of a restriction fragment of a gene for δ-endotoxin of B.thuringiensis, this part coding for the NH2-terminal part of the polypeptide active on the larvae of Lepidoptera, and from the 3′ part of this fragment coding for the COOH part of the polypeptide,
        the isolation of the hybrid fragment,
        its cloning in a vector, followed by its purification.
    
    


In an advantageous manner, the hybridization probes utilized are obtained from a gene for the δ-endotoxin derived from the strain aizawai 7-29 coding for a protein of 130 kDa, active against P.brassicae and inactive towards S.littoralis, this gene having been cloned in the recombinant plasmid pHTA2.
In an embodiment of the preceding procedure, the fragment recombined with the vector in the cloning step is elaborated from a HindIII-PstI restriction fragment derived from a single strain of B.thuringiensis, preferably aizawai 7-29. In particular, this fragment is carried preferentially by the recombinant plasmid pHTA6 such as isolated with the aid of a probe constituted by a PvuII fragment of 2 kb of the plasmid pBT15-88 corresponding to the internal part of a gene for the chromosomal crystal of the strain berliner 1715, starting from transforming clones containing nucleotide sequences derived from B.thuringiensis strains active against larvae of Lepidoptera, inter-alia of S.littoralis. 
In another embodiment of the invention, the fragment recombined with the vector in the cloning step is elaborated from several sequences of nucleotides derived from recombinant vectors containing sequences of nucleotides from at least two different strains of B.thuringiensis, possessing the same restriction maps and themselves containing all or part of the sequences of nucleotides capable of coding for a polypeptide active, in a preferential manner, against S.littoralis. 
In this case, the recombined fragment used in the cloning step is a fragment of about 3 kb, advantageously elaborated from a HindIII-HincII restriction fragment of about 1.1 kb derived from the entomocidus 6-01 strain and a HincII-PstI fragment of about 1.9 kb from the aizawai 7-29 strain. It corresponds to a truncated gene for δ-endotoxin.
The HindIII-HincII and HincII-PstI restriction fragments are carried more especially by the respective recombinant plasmids pHTE6 and pHTA6 such as isolated with the aid of the probe constituted by the PvuII fragment mentioned above.
The study of the toxicity towards the larvae of Lepidoptera of the bacterial strains modified with the aid of the sequences of nucleotides defined above, has made it possible to demonstrate their high toxic activity, in particular with regard to the caterpillars of S.littoralis. 
This activity was estimated from the point of view of the specificity index corresponding to the ratio
LC50 S.littoralis 
LC50 P.brassicae 
in which “LC50” represents the lethal concentration killing 50% of the larvae in 72 hours.
The utilization of such an index makes it possible to evaluate the activity of the bacterial strains studied without having to consider the level of expression of the polypeptides.
The results obtained, which are reported in the examples which follow, and the values of LD50 which are deduced from them, have shown that the bacterial strains modified according to the invention show a more specific toxic activity towards S.littoralis than the native crystal proteins of the strains aizawai 7-29 or berliner 1715.
Therefore, the invention relates to the use of these modified strains, of recombinant vectors containing the nucleotide sequences defined above, in particular the plasmid pHT671 and the plasmid pHT71, and these sequences themselves for the elaboration of larvicidal compositions preferentially toxic towards S.littoralis. 
The larvicidal compositions of the invention are thus characterized in that they contain an efficaceous quantity of polypeptides such as defined above or expressed by the nucleotide sequences mentioned above.
In order to produce these polypeptides the truncated genes for δ-endotoxin corresponding to the nucleotide sequences of the invention are advantageously implemented.
These genes can be used to produce in excess the toxic polypeptide in microorganisms permitting the expression of the above recombinant vectors. Suitable strains of microorganisms include E.coli or also B.subtilis. 
These truncated genes may be reintroduced into the strains of B.thuringiensis or into related species such as B.cereus, according to the standard techniques, for example, by transformation, conjugation or transduction. These techniques make it possible to produce the toxic polypeptide in large quantity without first having to modify the natural region of the promoter for the δ-endotoxin genes of B.thuringiensis. 
This transformation may be carried out by using methods derived from the transformation of the protoplasts of B.subtilis according to (11) or of the vegetative cells of B.thuringiensis as described in (12).
The introduction of recombinant plasmids by a system of the conjugation type may be carried out by using B.thuringiensis as host strain and B.subtilis or Streptococcus faecalis as strains of the donor type by operating according to (13) and (14).
As a variant, the sequences of nucleotides are introduced into microorganisms living in the environment or in association with the plants and capable of expressing recombinant vectors containing these sequences. The introduction may be carried out in microorganisms such as Pseudomonas by working according to the procedure described in (17), by the intermediary of plasmid vectors containing the transposon Tn5 and the gene for the toxin, or Azospirillum or Rhizobium by means of the intermediary of suicide vectors derived from the plasmid RP4 and of mobilizing plasmids functional in Gram negative bacteria (for example, pRK2013) according to the procedures described in (18).
The truncated genes are alone in the strains of Bacilli or, as a variant, are associated with different δ-endotoxin genes which makes it possible to obtain crystals synthesized by these strains specifically toxic towards given species of Noctuidae, or toxic both towards the Noctuidae and insects sensitive to other δ-endotoxins. These recombinations, carried out in vitro or in vivo with the nucleotide sequences of the invention and other δ-endotoxin genes showing different toxic specificities, lead to the construction of new genes coding for novel hybrid toxic proteins exhibiting a large spectrum of activity towards insects. These new genes and these novel proteins also enter into the framework of the invention.
In these applications, the nucleotide sequences of the invention may be transferred and expressed in plants sensitive to S.littoralis in order to diminish the devastation caused by these insects.
Among the plants to be protected, mention should be made of: cotton, clover, the tomatoe and alfalfa.
The transfer of the truncated gene into cotton plants may be carried out by transformation involving strains such as Agrobacterium as described in (15).
In addition, the invention relates to the plant cells, the plants and the seeds containing the nucleotide sequences defined above.
The plant cells according to the invention are cells, the genome of which after transformation by a non-essentially biological procedure possesses in a stable manner a sequence of nucleotides capable of expressing a polypeptide toxic towards S.littoralis, such as that defined above. The invention also relates to the plant cells derived from their division.
The plants according to the invention are plants transformed by a non-essentially biological procedure, having in particular as predator S.littoralis, the genome of which possesses in a stable manner a sequence of nucleotides such as that defined above, capable of expressing a polypeptide toxic towards S.littoralis. The plants in question are also plants derived from their reproduction, their multiplication or hybrid crosses.
In accordance with another feature, the invention relates to plants having in particular as predator S.littoralis, possessing in addition to their initial phenotypic and genotypic characters the property of expressing a polypeptide toxic preferentially towards S.littoralis, this property resulting from the insertion in their genome by means of genetic manipulation of a sequence of nucleotides capable of expressing the said polypeptide.
In addition, the invention relates to seeds capable of giving rise to a plant such as that defined above or derived from such a plant, characterized in that they have integrated into their genome by genetic manipulation a nucleotide sequence described above.



Other characteristics and advantages of the invention will become apparent in the course of the description and in referring to the examples in which:
FIG. 1 presents the restriction map of the plasmids pHTA6 and pHTE6,
FIG. 2, the restriction map of a gene for a crystal protein of the aizawai 7-29 strain cloned in the plasmid pHTA2 and defining the DNA fragments which are used as probe,
FIG. 3 shows the fragment of 6.6 kb cloned in pHTA6 and the result of a hybridization carried out between this fragment and the probes described in FIG. 2,
FIG. 4, the restriction map of the plasmid pHT671, and
FIG. 5, the photographs of the immunodiffusion tests.



The hybridization experiments carried out for the implementation of the invention were performed at 42° C. for 24 h in a solution containing 5×SSC, 30% formamide and 1 Denhardt (7) in the presence of the DNA probe labelled with 32P. The filters are washed at 42° C., 20 mn, by using successively the following solutions: 5×SSC in 30% formamide, 5×SSC, 2×SSC, 1×SSC and 0.5×SSC before drying at room temperature.
EXAMPLE 1
Construction of a DNA Sequence of about 3 kb Containing a Hybrid Gene of an Insecticidal Toxin.
This construction comprises:

    1/ the preparation of gene banks of B.thuringiensis 
    2/ the selection and characterization of transforming clones containing the genes of a crystal protein and nucleotide sequences responsible for the larvicidal activity,
    3/ in vitro recombination of these sequences in a cloning vector with construction of the plasmid pHT671.


These different steps are carried out as follows:
1/ Preparation of Gene Banks of B.thuringiensis. 
The total DNA of the aizawai 7-29 and entomocidus 6-01 strains of Bacillus thuringiensis is purified by using the method reported in (1) and 50 μg of each purified DNA are completely digested with the restriction enzyme PstI.
The DNA digested by PstI is analysed by horizontal electrophoresis on a 0.8% agarose gel and DNA fragments of a size of 5 to 8 kb are recovered from the agarose gels by electroelution in a manner described in (2).
The purified DNA fragments of 5-8 kb of the aizawai 7-29 strain are ligated to the DNA of the cloning vector pUC18 digested by PstI according to (3).
The purified DNA fragments of 5-8 kb of the entomocidus 6-01 chain are ligated to the DNA of the cloning vector pUC9 digested by PstI. The cells of E.coli JM83 are transformed with the ligation mixture as described in (4).
The transforming clones of E.coli are selected on LB medium containing 100 μg/ml of ampicillin.
2/ Isolation and Characterization of the Transforming Clones Containing the Genes for a Crystal Protein.


    A/ Screening of the transformed E.coli cells with the aid of an internal fragment of a gene of the crystal protein labelled with 32P, used as probe:


Transforming clones containing recombinant plasmids carrying the gene for the crystal are detected by colony hybridization according to the method described in (5), by using as probe a PvuII fragment of 2 kb of the pBT 15-88 plasmid corresponding to an internal part of the gene for the crystal protein located on the chromosome of the berliner 1715 strain.

    B/ Characterization of the recombinant plasmids present in the clones which react with the above probe.


Two recombinant plasmids, pHTA6 and pHTE6, isolated respectively from gene banks constructed from the strains aizawai 7-29 and entomocidus 6-01, show a homology with this probe. In each case, a DNA fragment of about 6.6 kb was cloned.
The restriction map of the two plasmids is given in FIG. 1. The comparison of the restriction sites shows that the two DNA fragments cloned appear to be identical.
In order to delimit the sequences corresponding to the gene for the δ-endotoxin, different DNA fragments labelled with 32P, derived from a gene of the crystal previously characterized, and cloned in the recombinant plasmid pHTA2, are utilized as probes. This latter gene for the crystal also derived from the aizawai 7-29 strain codes for a protein of 130 kd active against P.brassicae but not against S.littoralis. This type of gene possesses the same restriction map as the gene for the δ-endotoxin derived from the berliner 1715 strain. In FIG. 2 is shown the restriction map of this gene for the crystal protein of the aizawai 7-29 strain cloned in the plasmid pHTA2. The thick lines shown above the map correspond to the fragments used as hybridization probes.
The plasmids pHTA6 and pHTE6 are hydrolysed by different restriction endonucleases, analysed by horizontal electrophoresis on a 0.8% agarose gel and hybridized with the different probes.
The transfer of the DNA to nitrocellulose filters is carried out according to the method of SOUTHERN described in (6). The hybridization is conducted at 42° C. for 24 hours in a solution containing: 5×SSC, 30% formamide and a 1× Denhardt mixture described in (7) in the presence of a DNA probe labelled with 32P. The filters are then washed at 42° C. for 20 minutes, by using successively the following solutions: 5 SSC in 50% formamide, 5 SSC, 2 SSC, 1 SSC and 0.5 SSC before being dried at room temperature.
The results of these hybridization experiments are summarized in FIG. 3. It appears that each extremity of the cloned DNA fragments of 6.6 kb shows a homology with the probes. The PstI-KpnI fragment of 1.5 kb reacting with the probe No. 3 corresponds to the 3′ end of a gene of the crystal protein present in both the aizawai 7-29 and entomocidus 6-01 strains. As pointed out in FIG. 3, the probes No. 1 and No. 2 corresponding to the 5′ end of the gene for the δ-endotoxin of pHTA2 hybridize with the HindIII-HincII fragment of 1.1 kb contained in the plasmid pHTA6. The probe No. 3 which covers the 3′ end of the gene of the δ-endotoxin of pHTA2 hybridizes with the HindIII-PstI fragment of 0.4 kb contained in the plasmid pHTA6. It should be noted that a weak hybridization signal is obtained with the probe No. 2 whereas the two other probes give easily detectable signals.
From these results, the inventors have established that the HindIII-PstI DNA fragment of 3 kb corresponds to a large part of a gene for the δ-endotoxin which commences close to the central HindIII site. It seems clear in the light of results of the hybridization experiments that the gene for the δ-endotoxin shows substantial differences from those characterized in the prior art. On the basis of these results it was decided to clone the HindIII-PstI fragment of 3 kb in the vector pUC9.
3/ Construction of the Plasmid pHT 671 Containing a Hybrid Gene of the Reconstituted Insecticidal Toxin.
The HindIII-HincII DNA fragment of 1.1 kb derived from the plasmid pHTE6 and the HincII-PstI DNA fragment of 1.9 kb derived from the plasmid pHTA6 are purified on agarose gels.
Equal amounts of the two purified DNA fragments and the DNA of pUC9 digested with HindIII and PstI are mixed and ligated. The ligation mixture is used to transform competent cells of E.coli JM83, then the transformed E.coli cells are selected on LB medium containing ampicillin. One of the interesting recombinant clones examined contains a plasmid designated by pHT671, the restriction map of which was determined and is shown in FIG. 4. This plasmid (pHT671) contains a DNA fragment of 3 kb inserted in the vector pUC9. This DNA sequence has the same restriction map as the HindIII-PstI fragments of 3 kb contained in the plasmids pHTA6 and pHTE6, but corresponds to a reconstituted DNA molecule constructed by in vitro recombination from DNA sequences derived from the aizawai 7-29 strains on the one hand and entomocidus 6-01 on the other.
EXAMPLE II
Study of the Nucleotide Sequence of the Promoter Region and of the Region Coding for the NH2-terminal Part of the δ-endotoxin Active Against the Noctuidae.
The HindIII-HincII fragment of pHT671 is sequenced in conformity with the method described in (8) by using a M13 system. In order to obtain partially overlapping cloned DNA fragments which will be used in the sequencing of the DNA, recourse is had to the method of subcloning by deletion in M13, developed by DALE et al (9).
The sequence of 940 nucleotides of the HindIII-HincII fragment which has a length of about 1 kilobase corresponds to the chain arrangement I above.
The analysis of this sequence shows that the largest open reading frame starts at position 241 and that a potential site of binding to the ribosomes, GGAGG, is found six base pairs upstream from this ATG codon (position 230 to 235). The region localized between the nucleotides 137 and 177 (position −103 to −63 upstream from the ATG codon) is strongly homologous with the region present upstream from the gene for the crystal of the strain kurstaki HD1 Dipel (BTK) sequenced by WONG et al (1983) and described in (16) and the authors of which have shown that it contains three promoters BtI, BtII, and Ec, functional in B.thuringiensis and E.coli, respectively. The comparison between the amino acid sequences deduced from the first 750 nucleotides of the genes of BTK and pHT671, show that these polypeptides exhibit significant differences at the level of the N-terminal half of the active part derived from the protoxin (only 66% strict homology). It is important to note that it is the first time that a gene for the δ-endotoxin isolated from a strain active against the Lepidoptera codes for a polypeptide which shows substantial differences in this region. In fact, this N-terminal domain appears to be strongly conserved (more than 97% of strict homology) among all of the genes for the crystal active on Lepidoptera which have been sequenced hitherto. Moreover, the inventors have shown that the degree of variability is of the same order if the nucleotide sequences of pHT671 and other genes of the Lepidoptera type are considered.
EXAMPLE III
Construction of a DNA Sequence of about 2.7 kb Containing a Gene for a Larvicidal Toxin
In order to achieve this construction the DNA of the aizawai 7-29 strain of B.thuringiensis was used up to the step for the production of the plasmid pHTA6 as described in Example I.
The HindIII-PstI fragment of about 2.7 kb obtained from the plasmid pHTA6 was then subcloned in the vector pUC9, previously hydrolysed by the restriction enzymes HindIII-PstI in order to give the plasmid pHT71.
EXAMPLE IV
Study of the Sequence of Nucleotides Constituting the Plasmid pHT71 Coding for a Polypeptide Toxic Towards the Larvae of Lepidoptera of the Family of the Noctuidae
The HindIII-PstI fragment of 2.7 kb of pHTA6, which was subcloned in pHT71, was sequenced by means of the technique of Sanger et al. (8) using the phage M13 mp19 and the subcloning system by deletions developed by Dale et al (9). This system makes it possible to obtain M13 phages containing a series of partially overlapping DNA fragments which can be utilized for sequencing the DNA.
The sequence of nucleotides of this 2.7 kb fragment which corresponds to the chain arrangement (III) given above, was determined on the 2 DNA strands, with the exception of the last 212 nucleotides (position 2500 to 2711) which were sequenced only on a single strand.
The nucleotide sequence of this HindIII-PstI fragment has a length of 2711 nucleotides. This fragment contains the potential promoter as well as the largest part of the gene for the δ-endotoxin active on S.littoralis. 
EXAMPLE V
Study of the Specific Toxicity of the Recombinant Clones of E. coli JM83 (pHT671) and JM83 (pHT71) Against S.littoralis. 
The toxicity of the recombinant clones of E.coli JM83 containing pHT671 and of E.coli JM83 containing pHT71 was determined by biological tests on caterpillars of the P.brassicae and S.littoralis species as described by LECADET and MARTOURET in (10). The results were compared with the specific toxicity of the native crystal proteins purified from the strains berliner 1715 and aizawai 7-29, entomocidus 6-01 B.cereus 569 (containing the plasmid pBT45, pAMB1) against the two species of insects. The specific toxicity of the recombinant clone and of the strains of B.thuringiensis is expressed in terms of “specificity index” previously defined.
The results obtained are reported in table 1 below.
In this table, for E.coli strains, the concentration 1 corresponds to a 14 hours bacterial culture concentrated 20 times, disintegrated by ultrasound; for the B.thuringiensis strains the concentration is expressed in μg of crystal protein per μl of preparation. The toxic activity of the preparations was tested by the forced ingestion with 5 μl of preparation on caterpillars at the fifth stage of development, or by a technique of free ingestion utilizing larvae at the second stage of development.







TABLE 1







Comparative toxicity of a recombinant clone and two strains


of B. thuringiensis towards S. littoralis and P. brassicae.











S. littoralis
P. brassicae













LC50
LC50
LC50




2nd
5th
5th
Specificity index


Strains
larval
larval
larval
LC50 S. littoralis


and plasmids
stage
stage
stage
LC50 P. brassicae














JM83 (pUC18)
>1
>1
>1
—


JM83 (pHT671)
0.04
0.13
0.72
0.2


JM83 (pHTA2)
>1
>1
0.03
>30


JM83 (pHTA4)
>1
>1
>1
—


JM83 (pKT71)
ND
0.5
>1
<0.5


berliner 1715
ND
0.11
0.007
15.7


native crystals


aizawai 7.29
ND
0.02
0.04
0.5


native crystals


entomocidus 601
ND
0.028
0.012
2.3


native crystals


B. cereus 569
ND
0.38
0.054
7


(pBT45.pAMβ1)









Examination of the LC50 values summarized in this table 1 shows that the protein extracts of the recombinant clones JM83 (pHT671) and JM83 (pHT71) are preferentially toxic against S.littoralis. Secondly, a comparison of the values of the specificity index shows that the larvicidal activity of the recombinant clones is more specific by a factor of 2.5 times towards S.littoralis than the native crystal proteins of the aizawai strain. Moreover, the recombinant clones of JM83 (pHT671) and JM83 (pHT71) are very active against another insect of the family of the Noctuidae, Mamestra brassicae (in the case of the clone JM83 (pHT671) for example, the LC50 value is 0.02, utilizing larvae at the second stage of development).
These two results show that the gene for the larvicidal toxin constructed and cloned in the plasmids pHT671 and pHT71 codes for a protein specifically active against S.littoralis. 
Other preparations obtained from recombinant clones containing plasmids carrying genes coding for other types of δ-endotoxins (pHTA2 and pHTA4) are not active on S.littoralis: it may be seen that the plasmid pHTA2 codes for a δ-endotoxin specifically active on P.brassicae whereas the plasmid pHTA4 codes for a δ-endotoxin, the insect target for which has not yet been identified. It can also be seen that the crystalline inclusions produced by a strain of Bacillus cereus which has received the plasmid pBT45, one of the plasmids of the aizawai 7-29 strain which also carries a δ-endotoxin gene (the gene of plasmid origin of the aizawai 7-29 strain), are also specifically active on P.brassicae. 
Similar results are obtained by using, in the place of crude bacterial extracts, soluble protein extracts prepared from different recombinant clones of E.coli. 
On the basis of the LC50 values reported in the table above and a mean individual weight of 41 mg per L5 larva (fifth larval stage) of S.littoralis, the value of the LD50 was estimated at 2.4 μg/gram of larva for the native crystals of the aizawai 7-29 strain.
On these same bases and on the basis of equivalence factors making it possible to pass from the total bacterial mass to the quantity of specific proteins (about 2% of the total proteins in E.coli JM83 (pHT671), the LD50 corresponding to the toxin produced by the expression in E.coli JM83 of the gene according to the invention cloned in the plasmid pHT671, was determined and estimated at a value close to 5.5 to 6 μg/gram of larva.
On these same bases and after determination of the LC50 of soluble protein extracts prepared from ground cultures of E.coli JM83 (pHT671), the value of the LD50 corresponding to the toxin present in these extracts was estimated at 4.15 μg/gram of larva.
In the two cases and particularly in the case of the ground preparations of E.coli, the calculated values of LD50 are approximate and higher than that of the native crystals, because it is not a question of a purified toxin. However, these data indicate without ambiguity that the gene expressed by pHT671 specifies a δ-endotoxin exhibiting the specificity towards S.littoralis. In fact, the same type of estimation made with extract of E.coli JM83 (pHTA2) carrying a δ-endotoxin gene of different specificity leads to values 30 to 50 times higher than the LD50 of the soluble extracts towards S.littoralis (135 to 350 μg/gram of larva).
The foregoing data will easily make it possible for the person skilled in the art to develop active larvicidal compositions with the proteins of the invention.
Other toxicity experiments were carried out utilizing larvae of M.brassicae, S.frugiperda and S.littoralis at the second larval stage. The results obtained, expressed in terms of LC50 as defined for table 1, are given in table 2.







TABLE 2







ACTIVITY OF THE RECOMBINANT CLONES AGAINST


THE LARVAE OF INSECTS OF THE FAMILY OF THE


NOCTUIDAE: M. BRASSICAE, S. FRUGIPERDA, and


S. LITTORALIS.












INSECT LARVAE
M. BRASSICAE
S. FRUGIPERDA
S. LITTORALIS


STRAINS AND
AND
LC50
LC50
LC50


PLASMIDS
STAGE
2nd STAGE
2nd STAGE
2nd STAGE





JM 83 (pUC18)

NT
NT
NT


JM 83 (pHTA2)

>1
0.51
0.9


JM 83 (pHT671)

0.02
0.5
0.03


JM 83 (pHT71)

ND
ND
0.03


JM 83 (pHTA4)

>1
0.54
>1









It emerges from the examination of table 2 that the crude bacterial extracts of the recombinant clone JM83 (pHT671) are toxic towards M.brassicae and S.littoralis (the values of LC50 are 0.02 and 0.03, respectively) and weakly toxic towards S.frugiperda (LC50 of 0.5).
The extracts of the recombinant clone E.coli JM83 (pHTA2) are weakly active towards S.frugiperda and S.littoralis and not at all toxic towards M.brassicae. The extracts of the recombinant clone JM83 (pHTA4) are not toxic towards M.brassicae and S.littoralis and are weakly toxic toward S.frugiperda. 
These results confirm the high specific toxicity of the proteins obtained from pHT71 and pHT671 towards S.littoralis and show that this class of crystal protein is also very active towards M.brassicae. 
EXAMPLE VI
Study of the Specificity of the Polypeptides Expressed by the Clones Formed by Introduction of the Plasmids pHT671 and pHT71 into E.coli 
This study was carried out owing to immuno-diffusion tests. The results are reported in FIG. 5 (which includes FIGS. 5A and 5B).
The implementation of the immuno-diffusion experiment was done in conformity with the following protocol:
Soluble extracts of proteins of E.coli clones containing the plasmids pHT671, pHTA4, pHTA2 or pHT71, PUC18 were placed in the wells Nos. 2, 3, 4, 5, 6, respectively. A sample of a solubilized purified crystal of aizawai 7-29 was placed in the well No. 1 in order to serve as positive control.
In the test recorded in FIG. 5A an antiserum against all of the δ-endotoxins of aizawai 7-29, containing rabbit antibodies directed against the solubilized crystal proteins, was used and placed in the central well.
An immunoprecipitation line was observed in all of the cases except in the case of the extract of E.coli containing only the plasmid vector (well No. 6).
It was observed that the immuno-precipitation lines derived from the wells No. 4 and No. 5 cross, which shows that the products encoded by the plasmids pHTA2 and pHT71, respectively, display different antigenic determinants.
In the test recorded in FIG. 5B, the anti-serum used contained rabbit polyclonal antibodies against the crystal proteins of berliner 1715.
An immunoprecipitation line was observed with the extracts of E.coli JM83 (pHTA4) (well No. 3) JM83 (pHTA2) (well No. 4). On the other hand, the E.coli clones JM83 (pHT71) (well No. 5), JM83 (pHT671) (well No. 2) or JM83 (pUC9) (well No. 6) did not give immunoprecipitation.
It may be deduced from that that the genes for the crystal isolated in pHTA4 and pHTA2 express polypeptides having antigenic determinants in common with the proteins of the crystal of berliner 1715, a strain which is not specifically active towards S.littoralis. 
On the other hand, the crude extracts of E.coli containing the plasmids pHT671 and pHT71 contain polypeptides having antigenic determinants in common with the crystal proteins of the aizawai 7-29 strain, which are not related immunogenically with the crystal proteins of the berliner 1715 strain.
These results confirm those given previously with respect to the specificity of the genes isolated in the plasmids pHT71 and pHT671.
Antigen-antibody precipitation assays have made it possible to determine the level of expression of the δ-endotoxin genes in different recombinant clones.
The results obtained have shown that the crystal protein represents between 7 and 10% of the total cellular proteins of E.coli JM83 (pHTA2), between 2 and 3% in E.coli JM83 (pHT671) and between 0.5 and 1% in E.coli JM83 (pHTA4) and E.coli JM83 (pHT71).
The literature references cited in the examples are the following:

    (1) KLIER, A. F., LECADET, M-M. and DEDONDER, R., 1973, Sequential modifications of RNA polymerase during sporogenesis in Bacillus thuringiensis, Eur. J. Biochem., 36: 317-327.
    (2) MANIATIS, T., FRITSCH, E. F., SAMBROOK, J., 1982, Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, New-York
    (3) VIEIRA, J. and MESSING, J., 1982, The pUC plasmids, and M13mp7 derived system for insertion mutagenesis and sequencing with synthetic universal primers, Gene, 19: 259-268.
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## Claims

1. A recombinant expression and cloning vector comprising a nucleotide sequence coding for the N-terminal region of a polypeptide specifically toxic toward larvae of Lepidoptera of the family Noctuidae, wherein the nucleotide sequence consists of the sequence of an about 3 kb HindIII-PstI DNA fragment of the δ endotoxin gene of Bacillus thuringiensis, wherein the Bacillus thuringiensis is aizawai 7-29 strain.

2. A recombinant expression and cloning vector according to claim 1, wherein said nucleotide sequence hybridizes, at 42° C. in a solution containing 5×SSC, 30% formamide, and 1× Denhardt's, with a gene that expresses a polypeptide having the amino acid sequence of SEQ ID NO: 2 or larvicidal fragments thereof.

3. A recombinant expression and cloning vector according to claim 1, wherein the encoded polypeptide is capable of forming an immunological complex with antibodies directed against a polypeptide having the amino acid sequence of SEQ ID NO: 2 or larvicidal fragments thereof.

4. A modified bacterial strain comprising a nucleotide sequence coding for at least part of the N-terminal region of a polypeptide specifically toxic toward larvae of Lepidoptera of the family Noctuidae, wherein the nucleotide sequence consists of the sequence of an about 3 kb a HindIII-PstI DNA fragment of the δ endotoxin gene of Bacillus thuringiensis, wherein the Bacillus thuringiensis is aizawai 7-29 strain.

5. A modified bacterial strain according to claim 4, wherein said nucleotide sequence hybridizes, at 42° C. in a solution containing 5×SSC, 30% formamide, and 1× Denhardt's, with a gene that expresses a polypeptide having the amino acid sequence of SEQ ID NO: 2 or larvicidal fragments thereof.

6. A process for producing a polypeptide toxic towards Lepidoptera comprising:
(a) expressing the polypeptide in a microorganism capable of expressing recombinant vectors, wherein the recombinant vectors are at least one of: 
(i) a recombinant expression vector comprising a nucleotide sequence coding for the N-terminal region of a polypeptide specifically toxic toward larvae of Lepidoptera of the family Noctuidae, wherein the nucleotide sequence consists of the sequence of an about 3 kb HindIII-PstI DNA fragment derived from Bacillus thuringiensis var. aizawai 7-29; 
(ii) a recombinant expression vector comprising a nucleotide sequence coding for the N-terminal region of a polypeptide specifically toxic toward larvae of Lepidoptera of the family Noctuidae, wherein said nucleotide sequence hybridizes at 42° C. in a solution containing 5×SSC, 30% formamide, and 1× Denhardt's to a gene that expresses a polypeptide having the amino acid sequence of SEQ ID NO: 2 or larvicidal fragments thereof; or 
(iii) a recombinant expression vector comprising a nucleotide sequence coding for the N-terminal region of a polypeptide specifically toxic toward larvae of Lepidoptera of the family Noctuidae, wherein the encoded polypeptide is capable of forming an immunological complex with antibodies directed against a polypeptide having the amino acid sequence of SEQ ID NO: 2 or larvicidal fragments thereof; and 
(b) collecting the expressed polypeptide.

7. The process according to claim 6, wherein the recombinant vectors are introduced into microorganisms living in the environment or in association with plants.

8. The process according to claim 6 or 7, wherein the recombinant vectors are introduced into microorganisms in combination with different δ-endotoxin genes.

9. The process according to claim 6, wherein the microorganism is selected from the group consisting of E. coil, B. subtllis, B. cereus, or B. thuringiensis.

10. A process for obtaining a nucleotide sequence coding for a polypeptide toxic specifically toward Lepidoptera of the family Noctuidae, comprising:
(a) hybridizing a sequence of nucleotides from a strain of B. thuringiensis active against S. littoralis, and one or more hybridization probes, at 42° C. in a solution containing 5×SSC, 30% formamide, and 1× Denhardt's, wherein the hybridization probes are derived from nucleotide sequences comprising: 
(i) the 5′ part of a restriction fragment of a gene for the δ-endotoxin of B. thuringiensis that codes for the N-terminal part, amino acids 1-280 of SEQ ID NO: 2, of a polypeptide toxic toward Lepidoptera, or 
(ii) the 3′ part of a restriction fragment of a gene for the δ-endotoxin of B. thuringiensis coding for the COOH part, amino acids 621-1175 of SEQ ID NO: 2, of a polypeptide toxic toward Lepidoptera, 

(b) isolating at least one fragment, 
(c) cloning the fragment or fragments in a vector.

11. The process according to claim 10, wherein the hybridization probes utilized are obtained from a gene for δ-endotoxin derived from aizawai 7-29 strain for a protein of 130 kDa active against P. brassicae and inactive toward S. littoralis.

12. The process according to claim 10 or 11, wherein the fragment in (b) is one sequence of nucleotides derived from one strain of B. thuringiensis.

13. The process according to claim 12, wherein the fragment recombined with the vector in the cloning step (c) is a HindIII-PstI restriction fragment from the aizawai 7-29 strain.

14. The process according to claim 11, wherein the fragments recombined in step (c) are the insert of plasmid pHTA6 and the restriction fragments HindIII-HincII and HincII-PstI, which are the respective inserts of recombinant plasmids pHTE6 and pHTA6.

15. The process according to claim 10, wherein the fragment in (b) is at least 2 sequences of nucleotides from at least 2 different strains of B. thuringiensis possessing the same restriction maps and containing all or part of the sequences of nucleotides capable of coding for a polypeptide active toward S. littoralis.

16. The process according to claim 15, wherein the fragment recombined with the vector in the cloning step (c) is a HindIII-HincII restriction fragment from the entomocidus 6-01 strain and a HincII-PstI restriction fragment from the aizawai 7-29 strain.

17. A process for producing plants resistant to S. littoralis comprising
transforming a plant sensitive to S. littoralis with a recombinant vector of at least one of the following: 
(i) a recombinant expression vector comprising a nucleotide sequence coding for the N-terminal region of a polypeptide specifically toxic toward larvae of Lepidoptera of the family Noctuidae, wherein the nucleotide sequence consists of the sequence of an about 3 kb HindIII-PstI DNA fragment derived from Bacillus thuringiensis var. aizawai 7-29; 
(ii) a recombinant expression vector comprising a nucleotide sequence coding for the N-terminal region of a polypeptide specifically toxic toward larvae of Lepidoptera of the family Noctuidae, wherein said nucleotide sequence hybridizes at 42° C. in a solution comprising 5×SSC 30% formamide and 1× Denhardt's, to a gene that expresses a polypeptide having the amino acid sequence of SEQ ID NO: 2 or larvicidal fragments thereof; or 
(iii) a recombinant expression vector comprising a nucleotide sequence coding for the N-terminal region of a polypeptide specifically toxic toward larvae of Lepidoptera of the family Noctuidae, wherein the encoded polypeptide is capable of forming an immunological complex with antibodies directed against a polypeptide having the amino acid sequence of SEQ ID NO: 2 or larvicidal fragments thereof, 
wherein the transformed plant produces a polypeptide toxic toward S. littoralis.