Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-05-30
2004-02-03
Whisenant, Ethan (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S320100, C435S252300, C436S094000, C536S023710, C530S350000
Reexamination Certificate
active
06686149
ABSTRACT:
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.
BACKGROUND OF THE INVENTION
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 &dgr;-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 NH
2
-terminal half or N-terminus of the protein.
The studies carried out have shown the high specificity of the &dgr;-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 &dgr;-endotoxins hitherto identified do not code for a polypeptide preferentially active with regard to
S.littoralis.
SUMMARY OF THE INVENTION
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 &dgr;-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 &dgr;-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 NH
2
-terminal part of a &dgr;-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.
DETAILED DESCRIPTION OF THE INVENTION
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 GGG GTA GGA ACA TAA TCG GTC AAT TTT AAA
112
TAT GGG GCA TAT ATT GAT ATT TTA TAA AAT TTG TTA CGT TTT
172
TTG TAT TTT TTC ATA AGA TGT GTC ATA TGT ATT AAA TCG TGG
TAA TGA AAA ACA GTA TCA AAC TAT CAG AAC TTT GGT AGT TTA
232
ATA AAA AAA CGG AGG TAT TTT ATG GAG GAA AAT AAT CAA AAT
&ems
Dedonder Raymond
Lecadet Marguerite-Marie
Lereclus Didier
Martouret Daniel
Menou Ghislaine
Finnegan Henderson Farabow Garrett & Dunner L.L.P.
Institut Pasteur
Tung Joyce
Whisenant Ethan
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