Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Genetically modified micro-organism – cell – or virus
Reexamination Certificate
1992-02-18
2002-01-01
LeGuyader, John L. (Department: 1635)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Genetically modified micro-organism, cell, or virus
C435S069100, C435S252300, C435S320100, C536S023200, C536S023710, C536S024100
Reexamination Certificate
active
06335008
ABSTRACT:
BACKGROUND OF THE INVENTION
(1) Field of the Invention
The present invention relates to a hybrid gene incorporating a DNA fragment containing a gene coding for an insecticidal protein, more specifically a gene coding for an endotoxin active against Diptera. It also relates more particularly to recombinant vectors containing such gene or DNA fragment. It also relates to pro- and eukaryotic cells modified by these recombinant DNA vectors.
(2) Prior Art
The usefulness of
Bacillus thuringienses
endotoxins to control insect pests has been demonstrated over a wide range of crop and environmental pests.
Bacillus thuringienses
var.
israelensis
has been used as a biological insecticide to combat mosquito and black fly larvae in swamps, which are a real problem for human health, especially in tropical areas, and cause malaria and other diseases.
Commercial formulations consist of a culture of
Bacillus thuringiensis
var.
israelensis
bacterium in the sporulated stage consisting of spores and crystals. These crystals consist of proteins which have insect toxicity. These proteins act on the insect midguts when ingested by the larvae.
The main drawback of this approach is the fact that
Bacillus thuringiensis
bacterium is unstable in the environment (susceptible to U.V., washed away by intensive rains, etc.). Therefore one has to spray regularly which makes this method very expensive.
Strains of the Gram-positive bacterium
Bacillus thuriengiensis
(B.t.) produce intracellular protein crystals during the process of sporulation (Bulla et al. J. Bacteriol. 130, 375-383 (1977)). These crystal proteins, termed &dgr;-endotoxins, are toxic to a wide variety of Lepidoptera insects (Dulmage, et al., in Genetics and Relation to Insect Management (Hoy and Mekelvey, Jr. eds.), Rockefeller Foundation, New York, pp. 116-127 (1979)), some Diptera and Coleoptera. The endotoxins produced by different strains of B.t. may differ in their molecular structure and in their insect host range. In addition, one B.t. isolate may produce distinct types of crystal proteins.
Bacillus thuringiensis
var.
israelensis
(Goldberg-Margalit, Mosquito News 37, 353-358 (1977)), produces crystals that are highly toxic to larvae of mosquitos and black flies. In addition, the solubilized crystal proteins exhibit hemolytic activity and cytotoxicity towards mammalian cells (Thomas and Ellar, J. Cell Sci., 60, 181-197 (1983)).
B.t.
israelensis
crystals contain three main polypeptides of 130, 65 and 28 kDa with distinct antigenic properties. Controversy still exists on which component is responsible for the potent mosquitocidal activity in B.t.
israelensis
crystals. Originally, both insect toxicity and homolytic activity were attributed to the 28 kDa protein (Yamamoto, et al, Curr. Microbiol. 9, 279-284 (1983); Armstrong, et al, J. Bacteriol. 161, 39-46 (1985)). This was confirmed recently by molecular cloning and characterization of the B.t.
israelensis
gene encoding the 28 kDa crystal protein (Ward et al., FEBS, 175, 377-382 (1984); Ward and Ellar, J. Mol. Biol. 191: 1-11, 1986)). On the other hand, using purified crystal protein fractions, Visser et al. (Visser et al., FEMS Microbiol. Lett., 30, 211-214 (1986)) showed that, while the 28 kDa protein is hemolytic, the specific mosquitocidal activity resides entirely in the protein of 130 kDa.
A method has been described (McIntosh et al., in Molecular Form and Function of the Plant Genome; Plenum Press, New York, pp. 335-346 (1985)) for targeting insertions of foreign DNA into the chromosome of the Cyanobacterium Synechocystis 6803. This organism has a transformation system that enables it to take up exogenous DNA spontaneously. Donor DNA molecules were constructed by inserting a bacterial gene for kanamycin resistance into fragments of chromosomal DNA from the Cyanobacterium. Recipient cells were transformed to kanamycin-resistance with a frequency as high as four transformants per thousand cells. Analysis of DNA from transformants by transfer hybridization showed that the kanamycin-resistance gene was inserted in the cyanobacterial chromosome. Integration occurred by replacement of chromosomal DNA with homologous DNA that contained the foreign insert.
The ability of some Cyanobacterial species to take up exogenous DNA is central to the genetic modification. In many Cyanobacteria, DNA added to the growth medium enters cells by a naturally-occurring mechanism, as shown by using DNA isolated from spontaneous antibiotic-resistant mutants to transfer the resistant phenotype to sensitive cells (Shestakov and Khyen, Mol. Gen. Genet., 107, 372-375 (1970); Astier and Espardellier, C. R. Acad. Sci. Paris, 282, 795-797 (1976); Stevens and Porter, PNAS, USA, 77, 6052-6056 (1980); Griogoreiva and Shestakov, FEMS Microbiol. Lett. 13, 367-370 (1982)). This indicates that mutations in native Cyanobacterial genes can be introduced into wild-type cells. Cyanobacteria can also take up foreign DNA, as demonstrated by transformation with recombinant plasmids consisting of bacterial antibiotic-resistance genes linked to native Cyanobacterial plasmids (Buzby et al., J. Bacteriol. 154, 1446-1450 (1983); Van de Hondel et al., PNAS, USA, 77, 1570-1574 (1980)). In the cases, transformants were easily recovered on medium containing the appropriate antibiotics and were shown to harbor the recombinant plasmids. Another mechanism for DNA uptake, by conjugal transfer from
E. coli
cells, has been demonstrated recently with recombinant plasmids in a number of Cyanobacterial species (Wolk et al., PNAS, USA, 81, 1561-1565 (1984)). Whereas Cyanobacterial plasmids could be useful for complementation studies, they are less valuable for modifying genes resident on the chromosome.
In bacteria, plasmids have been used to construct insertion mutations in chromosomal genes (Ruvkun and Ausubel, Nature 289, 85-88 (1981)). This is accomplished by inserting an antibiotic resistance gene into a chromosomal gene that has been cloned in the plasmid, then the plasmid is introduced into wild-type cells to allow the antibiotic resistance gene to move from plasmid to chromosome by homologous recombination, finally recombinants are selected by curing cells of the plasmid while continuing to select for antibiotic resistance. This procedure has not been used in Cyanobacteria, in part because there is no efficient way to cure Cyanobacteria of autonomously replicating plasmids (Tandenau de Marsac et al., Gene, 20, 111-119 (1982)).
In an effort to develop a procedure for altering chromosomal genes in Cyanobacteria, Williams and Szalay (Williams and Szalay, Gene, 24, 37-51 (1983)) studied transformation in Synechococcus R2 using bacterial antibiotic resistance genes linked to fragments of Synechococcus R2 chromosomal DNA. It was found that the foreign DNA integrated efficiently into the Synechococcus R2 chromosome by homologous recombination and that, depending on the position of the resistance gene within the Cyanobacterial DNA, mutant transformants could be constructed (Williams and Szalay, Gene, 24, 37-51 (1983); and unpublished results, JGKW). These characteristics of the Synechococcus R2 transformation system indicate that it should be possible to introduce modified genes into the chromosome of this organism.
Experiments reported by McIntosh (McIntosh, L. et al, The Molecular Form and Function of the Plant Genome, Plenum Press, N. Y. 335-346 (1985)) show the Synchocystis 6803 is able to assimilate insertions of foreign DNA into its chromosome by homologous recombination, much as described in Synechococcus R2.
OBJECTS
It is one object of the invention to provide novel chimeric genes coding for a mosquitocidal protein, preferentially protein of
Bacillus thuringiensis.
Another object of the present invention is to provide novel hybrid plasmid vectors containing said chimeric genes, said vectors allowing the integration of said chimeric genes in the genome or remaining extrachromosomic.
A further object of the present invention is to provide Cyanobacteria transformed with said plasmids or said chimeric genes and a proce
Chungjatupornchai Wipa
McIntosh Lee
Vaeck Mark Albert
Board of Trustees operating Michigan State University
Larson Thomas G.
LeGuyader John L.
McLeod Ian C.
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