Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se
Reexamination Certificate
2000-12-21
2004-05-04
Nashed, Nashaat T. (Department: 1652)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
C435S189000, C435S468000, C435S410000, C435S320100, C435S252330
Reexamination Certificate
active
06730827
ABSTRACT:
FIELD OF THE INVENTION
This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding sulfate assimilation proteins in plants and seeds.
BACKGROUND OF THE INVENTION
Sulfate assimilation is the process by which environmental sulfur is fixed into organic sulfur for use in cellular metabolism. The two major end products of this process are the essential amino acids cysteine and methionine. These amino acids are limiting in food and feed; they cannot be synthesized by animals and thus must be acquired from plant sources. Increasing the level of these amino acids in feed products is thus of major economic value. Key to that process is increasing the level of organic sulfur available for cysteine and methionine biosynthesis.
Multiple enzymes are involved in sulfur assimilation. These include: High affinity sulfate transporter and low affinity sulfate transporter proteins which serve to transport sulfur from the outside environment across the cell membrane into the cell (Smith et al. (1995)
PNAS
92(20):9373-9377). Once sulfur is in the cell sulfate adenylyltransferase (ATP sulfurylase) (Bolchia et al. (1999)
Plant. Mol. Biol
. 39(3):527-537) catalyzes the first step in assimilation, converting the inorganic sulfur into an organic form, adenosine-5′ phospho-sulfate (APS). Next, several enzymes further modify organic sulfur for use in the biosynthesis of cysteine and methionine. For example, adenylylsulfate kinase (APS kinase), catalyzes the conversion of APS to the biosynthetic intermediate PAPS (3′-phospho-adenosine-5′ phosphosulfate) (Arz et al. (1994)
Biochim. Biophy. Acta
1218(3):447-452). APS reductase (5′ adenylyl phosphosulphate reductase) is utilized in an alternative pathway, resulting in an inorganic but cellularly bound (bound to a carrier), form of sulfur (sulfite) (Setya et al. (1996)
PNAS
93(23):13383-13388). Sulfite reductase further reduces the sulfite, still attached to the carrier, to sulfide and serine O-acetyltransferase converts serine to O-acetylserine, which will serve as the backbone to which the sulfide will be transferred to from the carrier to form cysteine (Yonelcura-Sakakibara et al. (1998)
J. Biolchem
. 124(3):615-621 and Saito et al. (1995)
J. Biol. Chem
. 270(27):16321-16326).
As described, each of these enzymes is involved in sulfate assimilation and the pathway leading to cysteine biosynthesis, which in turn serves as an organic sulfur donor for multiple other pathways in the cell, including methionine biosynthesis. Together or singly these enzymes and the genes that encode them have utility in overcoming the sulfur limitations known .to exist in crop plants. It may be possible to modulate the level of sulfur containing compounds in the cell, including the nutritionally critical amino acids cysteine and methionine. Specifically, their overexpression using tissue specific promoters will remove the enzyme in question as a possible limiting step, thus increasing the potential flux through the pathway to the essential amino acids. This will allow the engineering of plant tissues with increases levels of these amino acids, which now often must be added a supplements to animal feed.
SUMMARY OF THE INVENTION
The instant invention relates to isolated nucleic acid fragments encoding sulfate assimilation proteins. Specifically, this invention concerns an isolated nucleic acid fragment encoding an APS reductase and an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding an APS reductase. In addition, this invention relates to a nucleic acid fragment that is complementary to the nucleic acid fragment encoding APS reductase. An additional embodiment of the instant invention pertains to a polypeptide encoding all or a substantial portion of an APS reductase.
In another embodiment, the instant invention relates to a chimeric gene encoding an APS reductase, or to a chimeric gene that comprises a nucleic acid fragment that is complementary to a nucleic acid fragment encoding an APS reductase, operably linked to suitable regulatory sequences, wherein expression of the chimeric gene results in production of levels of the encoded protein in a transformed host cell that is altered (i.e., increased or decreased) from the level produced in an untransformed host cell.
In a further embodiment, the instant invention concerns a transformed host cell comprising in its genome a chimeric gene encoding an APS reductase, operably linked to suitable regulatory sequences. Expression of the chimeric gene results in production of altered levels of the encoded protein in the transformed host cell. The transformed host cell can be of eukaryotic or prokaryotic origin, and include cells derived from higher plants and microorganisms. The invention also includes transformed plants that arise from transformed host cells of higher plants, and seeds derived from such transformed plants.
An additional embodiment of the instant invention concerns a method of altering the level of expression of an APS reductase in a transformed host cell comprising:
a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding an APS reductase; and b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of altered levels of APS reductase in the transformed host cell.
An addition embodiment of the instant invention concerns a method for obtaining a nucleic acid fragment encoding all or a substantial portion of an amino acid sequence encoding an APS reductase.
REFERENCES:
EMBL Sequence Library Data Accession No.: D89631, Jul. 30, 1997, Sohlberg, L.E. et al., Nucleotide Sequence of a cDNA encoding a Cys proteinase from germinating bean cotyledons, XP-0021299910.
EMBL Sequence Library Data Accession No.: O49307, Ju. 1, 1998, Federspiel, N.A. et al., XP-002129911.
EMBL Sequence Library Data Accession No.: D25000, Nov. 30, 1993, Minobe, Y. et al., Rice cDNA from root, XP-002129912.
Frank W. Smith et al., PNAS, vol. 92:9373-9377, 9/1995, Plant members of a family of sulfate transporters reveal functional subtypes, XP-002129913.
Hideki Takahashi et al., Plant & Cell Phys., vol. 39 supp, pp.S148, 1998, Antisense repression of sulfate transporter in transgenicArabidopsis thalianaplants, XP-002121793.
Hideki Takahashi et al., PNAS, vol. 94:11102-11197, 9/1997, Regulation of sulfur assimilation in higher plants: A sulfate trnasporter induced in sulfate-starved roots plays a central role inArabidopsis thaliana.
EMBL Sequence Library Data Accession No.: X96761, Mar. 25, 1997, NG, A. et al., Isolation & characterization of a lowly expressed cDNA from the resurrection grassSporobolus stapfianuswith homology to eukaryote sulfate transporter proteins, XP-002121791.
EMBL Sequence Library Data Accession No.: AF016306, Jan. 8, 1998, Bolchi, A. et al., Coordinate modulation of maize sulfate permease and ATP sulfate permease and ATP sulfurylase mRNAs in response to variations in sulfur nutritional status: stereospecific down-regulation by L-cystein, XP-002121790.
EMBL Sequence Data Library Accession No.: O48889, Jun. 1, 1998, Bolchi, A. et al.
Frank W. Smith et al., The Plant Journal, vol. 12(4):875-884, Regulation of expression of a cDNA from barley roots encoding a high affinity sulphate transporter, XP-002129909.
Antje Prior et al., Biochimica et Biophysica Acta, vol. 1430:25-38, 1999, Structural and kinetic properties of adenylyl sulfate reductase fromCatharanthus roseuscell cultures.
Senta Heiss et al., Plant Mol. Biol., vol. 39:847-857, 1999, Cloning sulfur assimilation genes ofBrassica junceaL.: cadmium differentially affects the expression of a putative low-affinity sulfate transporter and isoforms of ATP sulfurylase and APS reductase.
John L. Wray et al., Chemico-Biological Interactions, vol. 109:153-167, 1998, Redefining reductive sulfate assimilation in higher plants: a role for APS reductase, a new member of the thioredoxi
E. I. du Pont de Nemours and Company
Nashed Nashaat T.
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