Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
2000-02-14
2003-04-29
McKelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Vector, per se
C435S325000, C536S023100, C536S024100
Reexamination Certificate
active
06555366
ABSTRACT:
The object of the invention is nucleotide sequences of bacteria, in particular Gram
+
bacteria such as bacteria of the Bacillus type and more particularly nucleotide sequences of the cryIIIA gene for the control of the expression of DNA sequences in a cell host.
The cryIIIA gene codes for a toxin specific for the Coleoptera and is weakly expressed by
Bacillus thuringiensis
when it is cloned in a low copy number plasmid.
Bacillus thuringiensis
is a Gram-positive bacterium which produces significant quantities of proteins in the form of crystals having a toxic activity towards insect larvae. Two groups of crystal proteins are known, based on the amino acid sequences and the toxicity specificities:
1) the class of the Cry toxins (I, II, III, etc . . . ) which have similar structures;
2) the class of the Cyt toxins, which is not related to the Cry class (Höfte, H et al. 1989, Microbiol. Rev. 53: 242-255)
These toxins of
B. thuringiensis
are of general interest for the purpose of the development of bio-pesticides and also in as much as the synthesis of crystal proteins is known to be perfectly co-ordinated with the sporulation phase of the organism, making this organism interesting for the study of genetic regulation in sporulating Gram-positive bacteria.
Various mechanisms implicated in the regulation of the synthesis of the crystal proteins of
B. thuringiensis
have been described. The high level of expression of these proteins is attributed, at least in part, to the stability of the mRNA. Some authors have attributed the stability of this mRNA to the presence downstream from the gene for the toxin of a structure playing a terminator role which might act as a positive retro-regulator by protecting the 3′ end of the mRNA from degradation by nucleases, thus increasing the half-life of the transcripts (Wong, H. C. et al., 1986 Proc. Natl. Acad. Sci. USA 83: 3233-3237).
A hypothesis has also been put forward concerning the presence of polypeptides implicated in the synthesis of crystal proteins, polypeptides which are supposed to act either by directing the folding of the protein in the form at a protein having a stable conformation or to protect these proteins from proteolytic degradation.
Studies with the electron microscope and biochemical studies of sporulation in
B. thuringiensis
show that the production of the crystal protein is dependent on sporulation and is located in the mother cell compartment (Ribier, J. et al. 1973 Ann. Inst. Pasteur 124A: 311-344).
Recently, two sigma factors, sigma 35 and sigma 28, which specifically direct the transcription of the cryIA genes have been isolated and characterized. These amino acid sequences exhibit an identity of 88 and 85% with the sigma factors E and K of
Bacillus subtilis
, respectively (Adams, L. F., 1991, J. Bacteriol. 173: 3846-3854). These sigma factors are produced exclusively in sporulating cells and are capable of functioning in the mother cell compartment, confirming that the expression of the genes for the crystal protein is controlled in time and space. Thus, in the prior art it has been concluded that the expression of the gene with time is, at least in part, ensured by the successive activation of the sigma factors specific for sporulation. Hitherto, three groups of promoters have been identified. Two of these groups include promoters recognized by specific sigma factors and, according to the prior art, the sigma factors associated with the third group of promoters (including that of the cryIIIA gene) have not been identified (Lereclus, D., et al. 1989 American Society for Microbiology, Washington, D.C.).
Finally, the copy number of the plasmid bearing the gene seems to be an important factor for the expression of the cry gene in
B. thuringiensis
. In the
B. thuringiensis
wild type strain, the cry genes are localized on large plasmids, present in a low number of copies.
Cloning experiments with a 3 kb HindIII fragment cloned in a low copy number plasmid lead to a low production of toxins in a non-crystal-forming strain (cry
−
) of
B. thuringiensis
. On the other hand, large quantities of toxins are synthesized when the gene is cloned in plasmids of high copy number (Arantes, O et al. 1991, Gene 108: 115-119).
SUMMARY OF THE INVENTION
The object of the invention is agents making it possible to obtain a high level of expression of the protein encoded in the cryIIIA gene and more generally agents making it possible to control the level of expression of DNA sequences coding for a specific protein of interest in bacterial strains, preferably Gram
+
strains such as Bacillus strains, since it is possible to obtain this expression when the coding DNA sequence is located on a vector, in particular on a plasmid of low copy number.
Generally speaking the invention relates to an expression system comprising a DNA sequence, able to intervene in the control of the expression of a coding nucleotide sequence and obtained by associating two distinct nucleotide sequences intervening in different but, preferably, not dissociable ways in the control of the expression of the coding sequence. The first nucleotide sequence exhibits a promoter activity whereas the second sequence, initiated by the promoter activity of the first, intervenes to enhance the expression of the gene. The DNA sequence of the invention makes it possible to attain a high level of expression of the coding part of a gene in a bacterium, in particular a Gram
+
type of bacterium.
The first nucleotide sequence of the expression system of the present invention identified in the framework of the present demand as being the promoter consists of either the promoter of the host strain in which the gene of interest to be expressed is introduced, or of an exogenous promoter, functional in the host used. The second nucleotide sequence of the expression system of the invention identified in the present application as being the “downstream region” designates any sequence preferably situated between the promoter and the sequence coding for a gene to be expressed, able to play a role particularly at the post-transcriptional level when the gene is expressed. More particularly, the downstream region does not act directly on the translation of the coding sequence to be expressed.
In a preferred manner, the “downstream region” consists of a nucleotide sequence, particularly an S2 sequence or a sequence analogous to S2, containing a region essentially complementary to the 3′ end of the RNA, particularly the 16S RNA, of the ribosomes of bacteria, particularly of Gram
+
bacteria of the Bacillus type.
The nucleotides forming the DNA sequence according to the invention may or may not be consecutive in the sequence from which the DNA sequence is defined.
In the context of the present application the expression “DNA sequence able to intervene in the control of the expression of a coding nucleotide sequence” expresses the capacity of this DNA sequence to initiate or prevent the expression of the coding sequence or to regulate this expression in particular at the level of the quantity of the product expressed.
A DNA sequence according to the invention is such that the coding nucleotide sequence that it controls is placed immediately downstream, in phase with the same reading frame as it or, on the other hand, it is separated from this DNA sequence by a nucleotide fragment.
Hence the invention relates to a DNA sequence for the control of the expression of a coding sequence for a gene in a cell host, the DNA sequence is characterized in that it includes a promoter and a nucleotide sequence or downstream region situated in particular downstream of the promoter and upstream of said coding sequence. The nucleotide sequence or downstream region contains a region essentially complementary to the 3′ end of a bacterial ribosomal RNA. The DNA sequence of the invention is capable of intervening to enhance the expression of the coding sequence placed downstream in a cell host.
The inventors have identified a DNA sequence of the type previously descri
Agaisse Herve
Lereclus Didier
Institut Pasteur
McKelvey Terry
Sandals William
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