Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of...
Patent
1995-12-20
2000-10-31
Brusca, John S.
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
4352523, 4353201, 435476, 536 231, 536 241, C12N 100, C12N 1574, C12N 1563, C07H 2104
Patent
active
061401048
DESCRIPTION:
BRIEF SUMMARY
The object of the invention is nucleotide sequences of bacteria, in particular Gram.sup.+ 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: structures; (Hofte, 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 coordinated 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 of 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.sup.-) 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,
REFERENCES:
Molecular Microbiology, vol. 4, No. 12, pp. 2087-2094, 1990, C. Dankocsik, et al., "Activation of a Cryptic Crystal Protein Gene of Bacillus Thuringiensis Subspecies Kurstaki by Gene Fusion and Determination of the Crystal Protein Insecticidal Specificity".
Molecular and General Genetics, vol. 214, pp. 365-372, 1988, William P. Donovan, et al., "Isolation and Characterization of EG2158, A New Strain of Bacillus Thuringiensis Toxic to Coleopteran Larvae, and Nucleotide Sequence of the Toxin Gene".
Journal of Bacteriology, vol. 175, No. 10, pp. 2952-2960, May 10, 1993, Marlene Teixeira De Souza, et al., "Full Expression of the cryIIIA Toxin Gene of Bacillus Thuringiensis Requires a Distant Upstream DNA Sequence Affecting Transcription".
Molecular Microbiology, vol. 13, No. 1, pp. 97-107, 1994, Herve Agaisse, et al., "Structural and Functional Analysis of the Promoter Region Involved in Full Expression of the cryIIIA Toxin Gene of Bacillus Thuringiensis".
Agaisse Herve
Lereclus Didier
Brusca John S.
Institut Nationale de la Recherche Agronomique
Institut Pasteur
Sandals William
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