Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
1997-01-03
2001-08-07
Smith, Lynette R. F. (Department: 1649)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C800S295000, C800S278000, C435S069100, C435S320100, C435S419000, C435S468000, C435S252300, C536S023200, C536S023600, C536S024100
Reexamination Certificate
active
06271442
ABSTRACT:
The invention relates to a method of producing pathogen-resistant plants, plants and plant components produced by the method, new DNA transfer vectors and DNA expression vectors and finally the use of a protein-synthesis inhibitor protein for producing pharmaceutical preparations.
BACKGROUND OF THE INVENTION
It is known for example from Ann. Rev. Plant Physiol. 1979, 30: 105-130 and Ann. Rev. Plant. Physiol. 1984, 35:34-275 that plants utilize a great variety of mechanisms to protect themselves from infections by pathogens. These mechanisms include for example modifications in the cell wall structure, synthesis of toxically acting phytoalexines, accumulation of so-called PR proteins (pathogenesis-related proteins), protease inhibitors and enzymes with hydrolytic functions.
It is further known for example from Biochem. J. 1983, 216:617-625 that various plants can generate proteins which have the ability of inhibiting the ribosomes of eucaryotes. Characteristic of such proteins inhibiting protein synthesis is the property of not influencing the plant-inherent ribosomes whilst they inactivate the plant-foreign ribosomes. Such proteins have become known in particular under the designation “RIP” proteins (ribosome-inhibiting proteins). Of most of these proteins, only their molecular weight and their mode of action are known.
Among the plants in which RIP proteins have been found are the barleys. Thus, in Carlsberg Res. Commun. Vol. 51,1986, p. 129-141, the purified protein, the molecular weight thereof and the amino acid sequence are described.
It is further known, for example from Biochemica et Biophysica Acta 880, 1986, p. 161-170 that RIP proteins are able to inhibit “in vitro” pathogens.
SUMMARY OF THE INVENTION
In the investigation of in particular barley plants the genes which encode for protein-synthesis inhibitors (PSI) have been identified. It has been found that these PSI genes encode for PSI proteins which can effectively block the protein synthesis of plant pathogens.
It has further been found that PSI genes isolated for example from barley plants can be fused with a great variety of active promotors, for example the wun1-promotor, which is described in detail in “The Plant Cell 1”, 1989, p.151-158 and that such promotor gene fusions can be incorporated into the genotype of plants and can produce transgenic plants which exhibit newly acquired pathogenic resistance.
It has further been found that the PSI protein can also be employed for producing pharmaceutical preparations which can be used to treat humans and animals affected by fungal, bacterial, viral or other pathogenic agents.
The PSI protein can be made in large amounts by introducing the PSI gene into bacterial overproducers. Purified PSI protein may be introduced in the form of infusion solutions into the blood path of humans or animals. The PSI protein inhibits the pathogen (for example AIDS viruses) without damaging the organism. The pathogen specificity of the PSI protein can possibly be further increased by coupling the PSI protein to pathogen-specific antibodies.
It is also possible to treat degenerate cells (cancer) in humans or animals by employing PSI protein. Thus, purified PSI protein or PSI protein which has been coupled to antibodies which detect specifically degenerate cells can be introduced into the blood path for destroying degenerate cells. Other forms of administration are possible, for example in capsules.
Suitable infusion solutions can be prepared by methods as usual and known for the preparation of aqueous infusion solutions.
Accordingly, the subject of the invention is a method of producing pathogen-resistant plants as is characterized in the claims, new DNA transfer vectors and DNA expression vectors as well as plants and plant components which can be obtained by the method according to the invention.
The subject of the invention is furthermore the use of the protein-synthesis inhibitor protein obtained by introduction of the protein-synthesis inhibitor gene into bacterial overproducers for the production of pharmaceutical preparations for the generation of pathogenic resistances, combatting pathogenic affection and/or degenerate cells.
Typical plant pathogens of which the protein synthesis can be inhibited by incorporation of a PSI gene are for example the fungi
Trichoderma reesei
and
Fusarium sporotrichioides.
(Attention is drawn to the review in M. Klinkowski, E. Mühle, E. Reinmuth and H. Bochow: “Phytopathology and plant protection I+II”, Akademie-Verlag, Berlin, 1974).
As is known, fusarium fungi attack mainly cereals and maize plants whilst fungi of the genus Trichoderma are to be found mainly on maize kernels.
It has surprisingly been found that the pathogen-inhibiting properties of PSI genes isolated for example from barley plants can also be transferred to plants of different species. Thus, it has been found for example that under the control of an active promotor, for example the wun1-promotor, a PSI gene isolated from barley can be incorporated into the genotype of tobacco plants. Tobacco plants which thereupon produce the PSI protein exhibit newly acquired resistance properties against for example the plant pathogenic
Rhizoctonia solani.
Rhizoctonia causes the so-called root-killer disease (affection of stem and root) in various plants, including potato and tobacco plants. Thus, newly acquired resistance properties in plants are directly correlated to the expression of PSI genes.
Apart from barley, protein-synthesis inhibitor genes can be isolated for example from all monocotyl and dicotyl plants and fused with a great variety of active promoters, for example pathogen-inducible promoters, constitutive promoters, development-specific promotors, organ-specific promotors and inducible promoters.
The following can be named as examples of plants into the genotype of which the new protein-synthesis inhibitor genes can be incorporated under the control of an active promotor:
all monocotyl useful plants, such as cereals
all dicotyl useful plants, such as solanaceae and cucurbitaceae.
The method according to the invention is thus particularly suitable for producing pathogen-resistant plants. However, through the expression of the protein-synthesis-inhibitor gene a resistance to insects, fungi, bacteria, viruses and viroids can also be achieved in humans and animals.
According to an advantageous further development, for executing the method according to the invention a protein-synthesis inhibitor gene is suitable which has the DNA sequence illustrated in FIG.
3
A. However, it will be apparent to the person skilled in the art that apart from this DNA sequence similar DNA sequences can be used to solve the problem set, for example a DNA sequence according to
FIG. 3B
which in the 5′ region has been completed by a corresponding cDNA clone.
The invention will be explained in detail hereinafter by way of example with reference to the isolation of a PSI gene from barley, fusion of said gene with an active promotor and transfer of the fusion product into the genotype of tobacco plants.
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patent: 5633442 (1997-05-01), Schell et al.
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Asano et al. Carlberg Res. Commun. 51, 129-141, 1986.*
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Sanchez-Serrano et al. (19
Jach Guido
Logemann Jurgen
Mundy John
Schell Jeff
Baker & Botts LLP
Haas Thomas
Max-Planck-Gesellschaft zur Forderung der Wissenschafen e.V.
Smith Lynette R. F.
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