Enhancer-increased gene expression in plants

Details Enhancer-increased gene expression in plants Enhancer-increased gene expression in plants

1998-08-18

2001-11-27

Nelson, Amy J. (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

C800S298000, C800S287000, C800S305000, C800S308000, C800S309000, C800S310000, C800S313000, C800S317000, C800S317100, C800S317200, C800S317300, C800S317400, C800S320000, C800S320100, C800S320200, C800S320300, C800S314000, C435S410000, C435S419000, C435S320100, C536S023100, C536S024100

Reexamination Certificate

active

06323393

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to increasing the expression of a gene, and in particular, to altering the activity of the promoter of the gene.
BACKGROUND OF THE INVENTION
Genetic manipulation depends on the introduction of chimaeric genes into plants and the expression of the introduced gene depends on the promoter. There are many reasons why it would be advantageous to have a method of improving the effectiveness of the promoter in these genes. Different promoters work with different efficiencies in different tissues. Different promoters work with different efficiencies in the same tissue, some, such as the Cauliflower Mosaic Virus 35S promoter (35S CaMV), are commonly considered to be a stronger promoter than the promoter from the nos gene. Promoters commonly consist of more that 1000 bp and when shortened work less efficiently. However, long sections of DNA produce technical difficulties in recombinant DNA techniques. Therefore, there are many instances when improved expression may be required. In experiments involving antisense, the highest expression possible might be required to achieve a commercial result. It is, therefore, an advantage to have DNA constructs available which would enhance, under the appropriate conditions, the expression of a given gene.
Sequences which activate transcription have been termed enhancers (Simpson et al. Nature (1986) 323, 551-554) and a sequence that is active as an enhancer has been obtained from the 35S promoter of CaMV (U.S. Pat. No. 5,164,316). The 35S promoter which contains this enhancer region is active in many plants and the promoter has been described as constitutive, acting in many tissues. However, while enhancer regions have been suggested for plant genes it has not been previously recognised that part of a plant promoter might have an enhancer activity in several different organs and in different species. For example, the −352 to −2 region of the pea RbcS gene was attached to the bacterial nos promoter and this gave strong light-induced expression in photosynthetic tissues. Similar experiments placing the element downstream of the coding sequence did not cause expression in tobacco (Fluhr et al, Science (1986) 232, 1106-1111).
The
pea PetE
gene was isolated by Last, D. I. and Gray, J. C. [Plant Molecular Biology (1989) 12, 655-666]. This gene encodes plastocyanin which is a 10 kDa copper protein involved in photosynthetic electron transfer. Thus, expression of this gene is required in organs such as leaves and stems in cells which contain chloroplasts. Deletion studies with the promoter region of this gene suggested that the promoter was active in leaves, stems and flowers, but not in roots, and that an element upstream from −784 to −992 repressed expression in leaves. Removal of this region produced a very ‘strong’ promoter (Pwee, K-H. and Gray, J. C. The Plant Journal (1993) 3 437-449).
BRIEF SUMMARY OF THE INVENTION
The invention is based on the surprising finding that a gene expressed in green photosynthetic tissue of pea has an enhancer region that is active in other species and in other tissues, including non-photosynthetic tissues.
It is an object of the present invention to provide a sequence of DNA which is active as an enhancer and causes an increase in expression of a promoter expressed in green tissues.
It is a further object of the present invention to provide a sequence of DNA which is active as an enhancer and causes an increase in expression of any promoter which is expressed in one or more of the roots, tubers, stems, leaves, flowers or seeds of plants.
It is also an object of the invention to provide a method of enhancing expression of genes in plants other than the plant from which the sequence was obtained.


REFERENCES:
patent: 5097025 (1992-03-01), Benfey et al.
patent: 0 278 659 A (1988-02-01), None
patent: 0 459 643 A (1991-09-01), None
Kim et al. Plant Molecular Biology. 1994. vol. 24: 105-117.*
EMBL Sequence Data Library, 1992, Accesion No. Z15046.
Dupree et al., 1991,The Plant Journal1:115-120.
Fisscher et al., 1994,Plant Molecular Biology26:873-886.
Last and Gray, 1989,Plant Molecular Biology12:655-666.
Pwee and Gray, 1993,The Plant Journal3:437-449.
Vorst et al., 1993,The Plant Journal4:933-945.

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