Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
2001-08-02
2002-10-15
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S183000, C435S410000, C435S419000, C435S252300, C435S320100, C435S180000, C435S325000, C435S254200, C435S440000, C536S023100, C536S023200, C536S023600, C800S295000, C800S278000
Reexamination Certificate
active
06465234
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 enzymes involved in the N-end rule pathway of protein degradation in plants and seeds.
BACKGROUND OF THE INVENTION
Cells continuously synthesize proteins from and degrade them into their component amino acids. The function of this seemingly wasteful process is twofold: first, to eliminate abnormal proteins whose accumulation would be harmful to the cell, and second, to permit the regulation of cellular metabolism by eliminating superfluous enzymes and regulatory proteins. The level of an enzyme depends on its rate of degradation as well as its rate of synthesis. Non-selective protein degradation occurs by a lysosomal mechanism. In eukaryotes a cytosolic ATP-dependent mechanism of protein degradation involves ubiquitin. This mechanism is based on the protein's half life which is partially determined by its N-terminal residue, giving this degradation mechanism the name of N-end rule pathway. The enzymes included in this application are involved in the N-end rule pathway of protein degradation.
Arginyl-tRNA-protein transferase (EC 2.3.2.8) is involved in catalyzing the post-translational conjugation of arginine to the amino termini of acceptor proteins. The function of these enzymes in eukaryotes is apparently to conjugate destabilizing amino acids to the amino termini of short-lived proteins. This reaction is a part of the N-end rule pathway of protein degradation.
Arabidopsis thaliana
encodes a single form of arginyl-tRNA-protein transferase while two forms, which are differentially spliced, are encoded by mice and humans (Kwon et al. (1999)
Mol. Cell. Biol.
19:182-193). A soybean EST encoding a peptide with similarities to cDNAs encoding arginyl-tRNA-protein transferase is found in the NCBI EST database having NCBI General Identifier No. 4313355.
The 26S proteosome is the central protease of the ubiquitin-dependent pathway of protein degradation. The 26S proteosome is formed by a barrel-shaped 20S core complex and two polar 19S complexes. The 20S particle contains the protease activity while the 19S complex contains isopeptidase, ATPase, and protein unfolding activities (Koster et al. (1995)
Mol. Biol. Rep.
21:11-20). Isopeptidases, also called ubiquitin carboxy-terminal hydrolases, belong to a family containing at least 10 different members. A 100 kDa de-ubiquitinating enzyme isolated from human tissues has been shown to contain “His and Cys domains” which are likely involved in the de-ubiquitinating activity, and an aspartic acid domain of unknown function Falquet et al. (1995)
FEBS Lett.
376:233-237. Because this enzyme cleaves peptide-linked and isopeptide-linked ubiquitin moieties from substrates, it is considered a de-ubiquitinase, instead of a isopeptidase (Falquet et al. (1995)
FEBS Lett.
359:73-77). An alignment of de-ubiquitinating enzymes shows that de-ubiquitinase is 99% similar to isopeptidase T. De-ubiquitinase contains a 23 amino acid insertion at position 629 of isopeptidase T which may account for the difference in activity between the two enzymes. Isopeptidase T is a monomeric ubiquitin-binding protein whose activity is inhibited by iodoacetamide and ubiquitin aldehyde (Hadari et al. (1992)
J. Biol. Chem.
267:719-727). A corn EST encoding a peptide with similarities to cDNAs encoding isopeptidase T is found in the NCBI database having NCBI General Identifier No. 4688518. Rice ESTs encoding peptides with similarities to cDNAs encoding isopeptidase T are found in the NCBI database having NCBI General Identifier Nos. 3768668 and 701567.
SUMMARY OF THE INVENTION
The instant invention relates to isolated nucleic acid fragments encoding enzymes involved in the N-end rule pathway of protein degradation. Specifically, this invention concerns an isolated nucleic acid fragment encoding an arginyl-tRNA-protein transferase or an isopeptidase T and an isolated nucleic acid fragment that is substantially similar to an isolated nucleic acid fragment encoding an arginyl-tRNA-protein transferase or an isopeptidase T. In addition, this invention relates to a nucleic acid fragment that is complementary to the nucleic acid fragment encoding arginyl-tRNA-protein transferase or isopeptidase T.
An additional embodiment of the instant invention pertains to a polypeptide encoding all or a substantial portion of an enzyme involved in the N-end rule pathway of protein degradation selected from the group consisting of arginyl-tRNA-protein transferase and isopeptidase T.
In another embodiment, the instant invention relates to a chimeric gene encoding an arginyl-tRNA-protein transferase or an isopeptidase T, or to a chimeric gene that comprises a nucleic acid fragment that is complementary to a nucleic acid fragment encoding an arginyl-tRNA-protein transferase or an isopeptidase T, 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 arginyl-tRNA-protein transferase or an isopeptidase T, 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 arginyl-tRNA-protein transferase or an isopeptidase T in a transformed host cell comprising: a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding an arginyl-tRNA-protein transferase or an isopeptidase T; 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 arginyl-tRNA-protein transferase or isopeptidase T 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 arginyl-tRNA-protein transferase or an isopeptidase T.
A further embodiment of the instant invention is a method for evaluating at least one compound for its ability to inhibit the activity of an arginyl-tRNA-protein transferase or an isopeptidase T, the method comprising the steps of: (a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding an arginyl-tRNA-protein transferase or an isopeptidase T, 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 arginyl-tRNA-protein transferase or isopeptidase T in the transformed host cell; (c) optionally purifying the arginyl-tRNA-protein transferase or the isopeptidase T expressed by the transformed host cell; (d) treating the arginyl-tRNA-protein transferase or the isopeptidase T with a compound to be tested; and (e) comparing the activity of the arginyl-tRNA-protein transferase or the isopeptidase T that has been treated with a test compound to the activity of an untreated arginyl-tRNA-protein transferase or isopeptidase T, thereby selecting compounds with potential for inhibitory activity.
BRIEF DESCRIPTION OF THE SEQUENCE LISTING
The invention can be more fully understood from the following detailed description and the accompanying Sequence Listing which form a part of this application.
Cahoon Rebecca E.
Falco Saverio Carl
Rafalski J. Antoni
Sakai Hajime
E. I. du Pont de Nemours and Company
Hutson Richard
Prouty Rebecca E.
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