Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide alters carbohydrate production in the plant
Reexamination Certificate
1999-06-15
2001-07-24
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide alters carbohydrate production in the plant
C800S278000, C800S286000, C800S293000, C800S298000, C800S306000, C800S312000, C800S314000, C800S320100, C800S320200, C800S320300, C800S322000, C800S281000, C800S320000, C435S069100, C435S419000, C435S468000, C435S320100, C536S023100, C536S023200, C536S023600, C536S024100, C536S024500
Reexamination Certificate
active
06265636
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to plant molecular biology. More specifically, it relates to nucleic acids and methods for modulating their expression in plants.
BACKGROUND OF THE INVENTION
The mitochondrial pyruvate dehydrogenase complex (mtPDC) catalyzes the decarboxylation of pyruvate yielding acetyl-CoA and NADH which are precursors of the Krebs cycle and oxidative phosphorylation. The mtPDC provides acetyl-CoA for the citrate synthase and acetyl-CoA hydrolase reactions and NADH for the electron transport system.
Pyruvate dehydrogenase complex (PDC) contains three primary component enzymes, pyruvate dehydrogenase (PDH, E1), dihydrolipoamide transacetylase (E2) and dihydrolipoamide dehydrogenase (E3).
The mtPDC provides a site for regulation due to its strategic metabolic location and the irreversible nature of the reaction. All PDCs exhibit product inhibition by acetyl-CoA and NADH. For mtPDCs, but not plastidial PDCs, reversible phosphorylation of the alpha subunit of E1 provides and “off-on” switch for mtPDC activity.
Reversible phosphorylation of PDC is catalyzed by two regulatory enzymes, PDH kinase (PDK) and P-PDH phosphatase. Both of these regulatory enzymes are specific for PDH.
SUMMARY OF THE INVENTION
It is the object of the present invention to provide nucleic acids and proteins relating to carbohydrate metabolism.
It is another object of the present invention to provide antigenic fragments of the proteins of the present invention.
It is another object of the present invention to provide transgenic plants comprising the nucleic acids of the present invention.
It is another object of the present invention to provide methods for modulating, in a transgenic plant, the expression of the nucleic acids of the present invention.
It is another object of the present invention to provide methods for manipulating cell metabolism in plants.
According to the present invention an isolated nucleic acid is provided comprising a member selected from the group consisting of
a) a polynucleotide that encodes a polypeptide of SEQ ID NO: 2 or 4;
b) a polynucleotide amplified from a
Zea mays
nucleic acid library using the primers of SEQ ID NOS: 5 and 6;
c) a polynucleotide comprising at least 25 contiguous bases of SEQ ID NOS: 1 or 3;
d) a polynucleotide having at least 60% sequence identity to SEQ ID NOS: 1 or 3, wherein the % sequence identity is based on the entire sequence and is determined by BLAST 2.0 under default parameters;
e) a polynucleotide comprising at least 25 nucleotides in length which hybridizes under low stringency conditions to a polynucleotide having the sequence set forth in SEQ ID NO: 1 or 3, wherein the conditions include hybridization with a buffer solution of 30% formamide, 1 M NaCl, 1% SDS at 37° C. and a wash in 2×SSC at 50° C.;
f) a polynucleotide complementary to a polynucleotide of (a) through (e).
In another aspect, the polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOS: 1 and 3.
In another aspect, the present invention relates to an isolated protein comprising a member selected from the group consisting of:
a) a polypeptide comprising at least 10 contiguous amino acids of SEQ ID NO: 2 or 4;
b) a polypeptide comprising at least 60% sequence identity to SEQ ID NO: 2 and 4, wherein the % sequence identity is based on the entire sequence and is determined by BLAST 2.0 using default parameters;
c) a polypeptide encoded by a nucleic acid of claim
1
; and
d) a polypeptide characterized by SEQ ID NO: 2 or 4.
In another aspect, the present invention relates to expression cassettes, comprising a nucleic acid as described, supra, operably linked to a promoter.
In another aspect, the nucleic acid is operably linked in antisense orientation to the promoter.
In another aspect, the present invention is directed to a host cell transfected with the expression cassette.
In another aspect, the present invention is directed to a method of modulating the level of PDK protein in a plant, comprising:
a) stably transforming a plant cell with a PDK polynucleotide operably linked to a promoter, wherein the polynucleotide is in sense or antisense orientation;
b) growing the plant cell under plant growing conditions to produce a regenerated plant; capable expressing the polynucleotide for a time sufficient to modulate PDK protein in the plant.
In yet another aspect, the present invention relates to a transgenic plant comprising an expression cassette comprising a plant promoter operably linked to any of the isolated nucleic acids of the present invention. In some embodiments, the transgenic plant is
Zea mays
. The present invention also provides transgenic seed from the transgenic plant.
In a further aspect, the present invention relates to a method of modulating expression of the genes encoding the proteins of the present invention in a plant, comprising the steps of (a) transforming a plant cell with an expression cassette comprising a polynucleotide of the present invention operably linked to a promoter; (b) growing the plant cell under plant growing conditions; and (c) inducing expression of the polynucleotide for a time sufficient to modulate expression of the genes in the plant. In some embodiments, the plant is maize. Expression of the genes encoding the proteins of the present invention can be increased or decreased relative to a non-transformed control plant.
Definitions
Units, prefixes, and symbols may be denoted in their SI accepted form. Unless otherwise indicated, nucleic acids are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. Numeric ranges are inclusive of the numbers defining the range. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. The terms defined below are more fully defined by reference to the specification as a whole.
By “amplified” is meant the construction of multiple copies of a nucleic acid sequence or multiple copies complementary to the nucleic acid sequence using at least one of the nucleic acid sequences as a template. Amplification systems include the polymerase chain reaction (PCR) system, ligase chain reaction (LCR) system, nucleic acid sequence based amplification (NASBA, Cangene, Mississauga, Ontario), Q-Beta Replicase systems, transcription-based amplification system (TAS), and strand displacement amplification (SDA). See, e.g.,
Diagnostic Molecular Microbiology: Principles and Applications
, D. H. Persing et al., Ed., American Society for Microbiology, Washington, D.C. (1993). The product of amplification is termed an amplicon.
The term “antibody” includes reference to antigen binding forms of antibodies (e.g., Fab, F(ab)
2
). The term “antibody” frequently refers to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof which specifically bind and recognize an analyte (antigen). However, while various antibody fragments can be defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments such as single chain Fv, chimeric antibodies (i.e., comprising constant and variable regions from different species), humanized antibodies (i.e., comprising a complementarity determining region (CDR) from a non-human source) and heteroconjugate antibodies (e.g., bispecific antibodies).
The term “antigen” includes reference to a substance to which an antibody can be generated and/or to which the antibody is specifically immunoreactive. The specific immunoreactive sites within the antigen are known as epitopes or antigenic determinants. These epitopes can be a linear array of monomers in a polymeric compositi
Miernyk Jan A.
Muszynski Michael G.
Randall Douglas D.
Sewalt Vincent J. H.
Thelen Jay J.
Fox David T.
Ibrahim Medina A.
Pioneer Hi-Bred International , Inc.
Pioneer Hi-Bred International Inc.
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