External regulation of gene expression by inducible promoters

Chemistry: molecular biology and microbiology – Treatment of micro-organisms or enzymes with electrical or... – Modification of viruses

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800205, 4353201, 536 241, C12N 1592, C12N 1511

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active

053647807

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BRIEF SUMMARY
FIELD OF THE INVENTION

This invention relates to the preparation and use of nucleic acid promoter fragments derived from several genes from corn, petunia and tobacco which are highly responsive to a number of substituted benzenesulfonamides and related compounds. Chimeric genes consisting of nucleic acid sequences encoding a desired gene product operably linked to one of these promoter fragments in recombinant DNA constructions may be made. Transformation of plants with such constructions will result in new plants in which the expression of the product encoded by such chimeric genes can be controlled by the application of a suitable inducing chemical.


BACKGROUND OF THE INVENTION

The ability to externally control the expression of selected genes and thereby their gene products in field-grown plants by the application of appropriate chemical substances in the field can provide important agronomic and foodstuff benefits. This control is especially desirable for the regulation of genes that might be placed into transgenic plants and has many applications including (1) prolonging or extending the accumulation of desirable nutritional food reserve in seeds, roots, or tubers, (2) producing and accumulating products in plant tissues at a defined time in the developmental cycle such that these products are convenient for harvest and/or isolation, and (3) initiating the expression of a pest-specific toxin at the site of pathogen attack. The latter example may provide a means of avoiding contamination of the ultimate food product with the toxic agent as well as minimizing the development of resistance in the pest population by selective, tissue specific, rather than constitutive expression of the toxic agent. These and other benefits have been unattainable to date since a practical means to bring known plant genes under external control in the field has not been available.
In eukaryotic systems, the expression of genes is directed by a region of DNA called the promoter. In general, the promoter is considered to be that portion of DNA in a gene upstream from the coding region that contains the site for the initiation of transcription. The promoter region also comprises other elements that act to regulate gene expression. These include the "TATA box" at approximately 30 bp (-30) 5' relative to the transcription start site and often a "CAAT box" at -75 bp. Other regulatory elements that may be present in the promoter are those that affect gene expression in response to environmental stimuli, such as light, nutrient availability, heat, anaerobioisis, the presence of heavy metals, and so forth. Other DNA sequences contained within the promoter may affect the developmental timing or tissue specificity of gene expression. In addition, enhancer-like sequences that act to increase overall expression of nearby genes in a manner that is independent of position or orientation have been described in a number of eukaryotic systems. Homologs of these enhancer-like sequences have been described for plants as well. The vast diversity of promoter function in eukaryotic systems therefore provides the opportunity to isolate promoters with relatively stringent requirements for their transcriptional activation which may be useful in regulating the timely expression of gene products in transgenic plants.
While current technology exists to transform plants with the genes encoding selected products, the expression of these genes is either continuous throughout the life cycle (controlled by a constitutive promoter), or regulated by the developmentally timed program of maturation inherent in each organ/tissue/cell (stage or tissue specific promoters) in which the gene product is destined to be expressed. Continuous expression precludes controlled production of a gene product at particular stages of the life cycle, in specific tissues or in response to environmentally unpredictable events. In addition, such constitutive expression could place a major penalty on yield, due to greatly increased energy demands accompanying prolonged high level

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