Chemistry: molecular biology and microbiology – Plant cell or cell line – per se ; composition thereof;... – Plant cell or cell line – per se – contains exogenous or...
Reexamination Certificate
1999-07-02
2001-06-05
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Plant cell or cell line, per se ; composition thereof;...
Plant cell or cell line, per se, contains exogenous or...
C435S410000, C435S006120, C435S069100, C435S189000, C435S252300, C536S023200
Reexamination Certificate
active
06242256
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 ornithine biosynthetic enzymes in plants and seeds.
BACKGROUND OF THE INVENTION
Ornithine is converted into arginine in the urea cycle. Intermediaries in the ornithine biosynthesis pathway are important in other steps of this cycle. Amino acid N-acetyl transferase (EC 2.3.1.1) catalyzes the first reaction in a pathway that leads to the synthesis of ornithine from L-glutamate giving N-acetylglutamate as its intermediary product.
Carbamoyl phosphate synthase I, the mitochondrial enzyme that catalyzes the first committed step of the urea cycle, is allosterically activated by N-acetyl glutamate. The rate of urea production by the liver is, in fact, correlated with the N-acetylglutamate concentration. Increased urea synthesis is required when amino acid breakdown rates increase, generating excess nitrogen that must be extracted. Increase in these breakdown rates are signaled by an increase in glutamate concentration through transamination reaction. This situation, in turn, causes an increase in N-acetylglutamate synthesis, stimulating carbamoyl phosphate synthetase and the entire urea cycle.
N-acety lglutamate kinase (EC 2.7.2.8) catalyzes the conversion of N-acetyl-L-glutamate and ATP into N-acetyl-L-glutamate-5-phosphate and ADP. N-acetyl-gamnma-glutamyl-phosphate reductase (EC 1.2.1.38) catalyzes the convertion of N-acetyl-L-glutamate 5-phosphate and NADPH to orthophosphate, NADP and N-acetyl-L-glutamate-5-semialdehyde. This activity is encoded by the argC locus in bacteria and
Synechocystis
. To date this gene has not been described in plants.
N-2-Acetyl-L-ornithine and L-glutamate are converted to ornithine in the presence of glutamate N-acetyl transferase (EC 2.3.1.35), also called ornithine acetyltransferase. This enzyme is encoded by the argJ locus in bacteria and
Synechocystis
. This enzyme is active in the mitochondrial matrix as a heterodimer consisting of two subunits processed from the same precursor protein.
SUMMARY OF THE INVENTION
The instant invention relates to isolated nucleic acid fragments encoding ornithine biosynthetic enzymes. Specifically, this invention concerns an isolated nucleic acid fragment encoding a N-acetyl-gamma-glutamyl phosphate reductase or a glutamate N-acetyl transferase and an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding a N-acetyl-gamma-glutamyl phosphate reductase or a glutamate N-acetyl transferase. In addition, this invention relates to a nucleic acid fragment that is complementary to the nucleic acid fragment encoding N-acetyl-gamma-glutamyl phosphate reductase or glutamate N-acetyl transferase.
An additional embodiment of the instant invention pertains to a polypeptide encoding all or a substantial portion of an ornithine biosynthetic enzyme selected from the group consisting of N-acetyl-gamma-glutamyl phosphate reductase and glutamate N-acetyl transferase.
In another embodiment, the instant invention relates to a chimeric gene encoding a N-acetyl-gamma-glutamyl phosphate reductase or a glutamate N-acetyl transferase, or to a chimeric gene that comprises a nucleic acid fragment that is complementary to a nucleic acid fragment encoding a N-acetyl-gamma-glutamyl phosphate reductase or a glutamate N-acetyl transferase, 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 a N-acetyl-gamma-glutamyl phosphate reductase or a glutamate N-acetyl transferase, 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 a N-acetyl-gamma-glutamyl phosphate reductase or a glutamate N-acetyl transferase in a transformed host cell comprising: a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a N-acetyl-gamma-glutamyl phosphate reductase or a glutamate N-acetyl transferase; 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 N-acetyl-gamma-glutamyl phosphate reductase or glutamate N-acetyl transferase 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 a N-acetyl-gamma-glutamyl phosphate reductase or a glutamate N-acetyl transferase.
A further embodiment of the instant invention is a method for evaluating at least one compound for its ability to inhibit the activity of a N-acetyl-gamma-glutamyl phosphate reductase or a glutamate N-acetyl transferase, the method comprising the steps of: (a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a N-acetyl-gamma-glutamyl phosphate reductase or a glutamate N-acetyl transferase, operably linked to suitable regulatory sequences; (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 N-acetyl-gamma-glutamyl phosphate reductase or glutamate N-acetyl transferase in the transformed host cell; (c) optionally purifying the N-acetyl-gamma-glutamyl phosphate reductase or the glutamate N-acetyl transferase expressed by the transformed host cell; (d) treating the N-acetyl-gamma-glutamyl phosphate reductase or the glutamate N-acetyl transferase with a compound to be tested; and (e) comparing the activity of the N-acetyl-gamma-glutamyl phosphate reductase or the glutamate N-acetyl transferase that has been treated with a test compound to the activity of an untreated N-acetyl-gamma-glutamyl phosphate reductase or glutamate N-acetyl transferase, thereby selecting compounds with potential for inhibitory activity.
BRIEF DESCRIPTION OF THE SEQUENCE LISTINGS
The invention can be more fully understood from the following detailed description and the accompanying Sequence Listing which form a part of this application.
Table 1 lists the polypeptides that are described herein, the designation of the cDNA clones that comprise the nucleic acid fragments encoding polypeptides representing all or a substantial portion of these polypeptides, and the corresponding identifier (SEQ ID NO:) as used in the attached Sequence Listing. The sequence descriptions and Sequence Listing attached hereto comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. §1.821-1.825.
TABLE 1
Ornithine Biosynthetic Enzymes
SEQ ID NO:
Clone
(Nucleo-
(Amino
Protein
Designation
tide)
Acid)
Jerusalem artichoke N-acetyl-
hel1.pk0002.h8
1
2
gamma-glutamyl phosphate
reductase
Corn N-acetyl-gamma-
Contig of:
3
4
glutamyl phosphate reductase
cco1.pk0046.h7
ceb1.pk0026.g8
cr1n.pk0185.a9
p0003.cgpfk13r
p0044.cjraf16r
p0128.cpiar56r
Rice N-acetyl-gamma-
rr1n.pk001.h10
5
6
glutamyl phosphate reductase
Soybean N-acetyl-gamma-
ses4d.pk0004.e10
7
8
glutamyl phosphate reductase
Wheat N-acetyl-gamma-
wlm96.pk037.f18
9
10
glutamyl phosphate reductase
Corn glutamate N-acetyl
Contig of:
11
12
transferase
p0004.cb1ec29r
p0016.ctsav50r
p0032.crcag34r
p0080.cgab
Cahoon Edgar B.
Cahoon Rebecca E.
Hitz William D.
Rafalski J. Antoni
E. I. Du Pont de Nemours and Company
Hutson Richard
Prouty Rebecca E.
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