Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-02-11
2002-12-03
Bui, Phuong T. (Department: 1638)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S006120, C435S410000, C435S419000, C435S320100, C536S023100, C536S024300, C536S024330, C800S278000, C800S295000
Reexamination Certificate
active
06489462
ABSTRACT:
BACKGROUND OF THE INVENTION
One of the primary goals of plant genetic engineering is to obtain plants having improved characteristics or traits. These characteristics or traits include virus resistance, insect resistance, herbicide resistance, enhanced stability and improved nutritional value, to name a few. Recent advances in genetic engineering have enabled the incorporation of preselected genes into plant cells to impart the desired qualities to the plant of choice. The introduced gene, i.e., “transgene,” is then expressed in the cells of the regenerated plant, so that the plant will exhibit the trait or characteristic encoded by the transgene.
To express a transgene in a plant cell, the proper regulatory signals must be present and in the proper location with respect to the transgene. These regulatory signals generally include a promoter region, a 5′ non-translated leader sequence and a 3′ polyadenylation sequence. The promoter region influences the rate at which the RNA product of the transgene, and resultant protein product of the transgene, is made. Promoter activity also can depend on the presence of several other cis-acting regulatory elements which, in conjunction with cellular factors, determine strength, specificity, and transcription initiation site (for a review, see Zawel and Reinberg,
Curr. Opin. Cell Biol
., 4, 488 (1992)). Strong promoters are able to direct RNA synthesis at a higher rate relative to weak promoters. Constituitive promoters direct RNA production in many or all cell types.
The cauliflower mosaic virus 35S promoter (CaMV35S) is a strong, constitutive promoter in plants (Odell et al.,
Nature
, 313, 810 (1985); Jensen et al.,
Nature
, 321, 669 (1986); Jefferson et al.,
EMBO J
., 6, 3901 (1987); Kay et al.,
Science
, 236, 1299 (1987); Sanders et al.,
Nucl. Acids Res
., 4, 1543 (1987)). This had been shown by detecting substantial levels of reporter gene proteins or mRNAs in extracts prepared from the leaves, stems, roots and flowers of transgenic plants. As a result, the CaMV35S promoter is widely used in the field of plant genetic engineering. Although the CaMV35S promoter appears to be a strong, constitutive promoter in assays involving cell extracts, detailed histological analysis of reporter gene products detectable at the cell and tissue level shows a rather high degree of variability of expression of the gene products in tissues of plants.
CaMV is a caulimovirus, a subgroup of pararetroviruses that has icosahedral capsids and infects only dicots, although the CaMV35 S promoter is a strong promoter in monocots. Sugarcane bacilliform virus (ScBV), Commelina yellow mottle virus (CoYMV) and rice tungro bacilliform virus (RTBV) are badnaviruses, a subgroup of pararetroviruses that have bacilliform capsids and infect mainly monocots. A promoter fragment isolated from CoYMV confers a tissue-specific pattern of expression that is different than the pattern conferred by the CaMV35S promoter. Transformed tobacco plants containing the CoYMV promoter linked to the beta-glucuronidase reporter gene (“GUS”; uidA) showed that while the CoMYV promoter is active in all organs, beta-glucuronidase activity occurs primarily in the phloem, the phloem-associated cells, and the axial parenchyma of roots, stems, leaves, and flowers (Medberry et al.,
Plant Cell
, 4, 185 (1992); Medberry and Olszewski,
Plant J
., 3, 619 (1993)). In contrast, the CaMV35S promoter is active in most cell types (Medberry et al.,
Plant Cell
, 4, 185 (1992); Medberry and Olszewski,
Plant J
., 3, 619 (1993)). Moreover, the CoYMV promoter is 30% as active in tobacco suspension cells and up to 25% as active in maize suspension cells compared to a duplicated CaMV35S promoter (Medberry et al.,
Plant Cell
, 4, 185 (1992)).
Transgenic rice containing the RTBV promoter linked to the GUS gene showed strong phloem-specific promoter activity. This was consistent with the expression of this promoter in rice protoplasts. However, the RTBV promoter showed only weak activity in maize protoplasts (Bhattacharyya-Pakasi et al.,
Plant J
., 4, 71 (1993); Yin et al.,
Plant J
., 7, 969 (1995)). In contrast, the corresponding CaMV promoter shows strong promoter activity in protoplasts and in almost all tissues of transgenic plants (reviewed by Hohn and Fütterer,
Curr. Opin. Genet. Dev
., 2, 90 (1992)).
Thus, what is needed is a highly expressed, constitutive promoter to express transgenes in fertile transgenic monocot and dicot plants.
SUMMARY OF THE INVENTION
The present invention provides an isolated and purified DNA molecule comprising a preselected DNA segment comprising a sugarcane bacilliform virus (ScBV) promoter, or a biologically active subunit thereof, that confers constitutively high levels of expression of operably linked preselected DNA segments in both monocot and dicot plants, plant tissue, plant parts or plant cells. While the nucleotide sequence of the genome of ScBV is known (Bouhida et al.,
J. Gen. Virol
., 74, 1 (1993)), the location of a promoter for genomic length viral RNA was not apparent, even after nucleotide sequence comparisons of the ScBV genome with promoter sequences of closely related viruses, such as CoYMV and RTBV. Surprisingly, the ScBV promoter is a strong and constituitive promoter in many cell types, unlike the strong tissue specific expression observed for CoMYV and RTBV promoters. A preferred embodiment of the invention is a preselected DNA segment comprising a ScBV promoter comprising SEQ ID NO:3, i.e., a preselected DNA segment that corresponds to nucleotide positions 5999-7420 of the ScBV genome. As described herein below, the ScBV promoter confers constitutive and vascular gene expression in
A. sativa
and
A. thaliana
. Thus, the ScBV promoter can be employed for constitutive or tissue-specific plant and non-plant gene expression in monocots and dicots. For example, the ScBV promoter may be linked to genes which confer to crops resistance against pests which attack crop grasses at the stem, e.g., aphids which vector barley yellow dwarf virus, or to confer tissue-specific resistance to dicotyledonous hosts, in which organs such as the roots are targeted by pathogens, e.g., soybean cyst nematode.
As used herein, “ScBV” includes any non-enveloped, bacilliform, DNA-containing badnavirus capable of systemically infecting Saccharum or related genera. Other distinguishing features of badnaviruses are described by Lockhart and Olszewski, in
The Encyclopedia of Virology
, Webster and Granoff (eds.), Academic Press, New York, N.Y. (1994)).
As used herein, the term “ScBV promoter” means a nucleotide sequence which, when that sequence is operably linked to a preselected DNA segment that encodes a protein, RNA transcript, or mixture thereof, results in the expression of the linked preselected DNA segment, i.e., the encoded RNA and/or protein. A preferred ScBV promoter has at least about 60%, preferably at least about 80%, more preferably at least about 90%, and even more preferably at least about 95%, nucleotide sequence identity to SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5. Another preferred embodiment of the invention is a ScBV promoter which comprises the minimum number of contiguous nucleotides which initiate RNA transcription.
As used herein, “biologically active” means that the promoter has at least about 0.1%, preferably at least about 10%, and more preferably at least about 25%, the activity of the ScBV promoter comprising SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5. The activity of a promoter can be determined by methods well known to the art. For example, see Medberry et al.,
Plant Cell
, 4, 185 (1992); Medberry et al., The
Plant J
., 3, 619 (1993); Sambrook et al., In:
Molecular Cloning: A Laboratory Manual
(1989); McPherson et al., U.S. Pat. No. 5,164,316.
Further provided is an expression cassette comprising a first preselected DNA segment comprising a ScBV promoter functional in a host cell, operably linked to a second preselected DNA segment encoding a protein, RNA transcript, or a combination thereof. A preferred host cell is a plant cell,
Lockhart Benham
Olszewski Neil
Somers David A.
Torbert Kimberly A.
Tzafrir Iris
Bui Phuong T.
Regents of the University of Minnesota
Schwegman Lundberg Woessner & Kluth P.A.
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