Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or...
Reexamination Certificate
1999-11-04
2004-02-24
Bui, Phuong T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
C435S006120, C435S069100, C435S183000, C435S410000, C435S419000, C435S252300, C435S320100, C530S370000, C536S023100, C536S023200, C536S023600, C536S024100, C536S024300, C536S024330, C536S024500, C800S295000
Reexamination Certificate
active
06696619
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 aminoacyl-tRNA synthetases in plants and seeds.
BACKGROUND OF THE INVENTION
Aminoacyl-tRNA Synthetases (AARS) are enzymes that charge (acylate) tRNAs with amino acids. These charged aminoacyl tRNAs then participate in mRNA translation and protein synthesis. The AARS show high specificity for charging a specific tRNA with the appropriate amino acid, for example valyl-tRNA with valine by valyl-tRNA synthetase or tryptophanyl-tRNA with tryptophan by tryptophanyl-tRNA synthetase. In general, per organism there are at least one AARS for each of the twenty amino acids. There are exceptions however. AARS are ancient enzymes, having functioned in translation since early life evolution. Some have speculated that the earliest aminoacyl-tRNA synthetases were mRNAs, not proteins, with the proteinaceous AARS described here emerging later (Neidhardt et al., (1975)
Annu. Rev. Microbiol
. 29:215-250). AARS are structurally diverse, although AARSs for some amino acids are more closely related than for others. AARSs are generally divided into two classes, class I and class II based on structural similarity and amino acid preferences (Eriani et al., (1990)
Nature
347:203-206).
Plants like all other cellular organisms have aminoacyl-tRNA synthetases. However, a full description of the plant ‘complement’ of aminoacyl-tRNA synthetases has not yet been described. Full-length cDNA, genomic clones, and EST sequences for a variety of plant aminoacyl-tRNA synthetases are known. However, several anticipated aminoacyl-tRNA synthetases have not been discovered.
Because of the central role of protein synthesis in life, any agent that inhibits or disrupts this activity is likely to be toxic. Aminoacyl-tRNA synthetases play a critical role in protein translation by linking genetic nucleic acid information to protein synthesis. Aminoacyl-tRNA synthetases perform this role by “reading” the identity of the different tRNAs and acylating them with the correct cognate amino acid. A large volume of research over several decades has been focused on identifying inhibitors of this process. Inhibitors of aminoacyl-tRNA synthetases have been found to be cytotoxic due to their inhibition of protein synthesis. As such they therefore could be used as herbicides or in aminoacyl-tRNA synthetase selectable marker systems (Lloyd et al., (1995)
Nucleic Acid Research
23(15):2882-2892). The genes disclosed herein can serve as the basis for testing whether the encoded aminoacyl-tRNA synthetases are sensitive to known inhibitors or other chemicals.
Biochemical processes are often compartmentalized in regions of cells, such as mitochondria, plastids, and lysosomes. These organelles are key sites for many biochemical pathways. Bioengineering of these processes may require targeting protein products to specific organells. One method to accomplish this involves the addition of an N-terminal prosequence (transit peptide) that directs protein entry into a specific organelle(s). Upon or shortly after transport into the organelle the transit peptide is usually proteolytically removed, and the mature protein is then able to function.
A few plant transit peptides have been shown empirically to be capable of directing fused proteins into specific organelles. However this ability appears to depend upon the structure of the protein being imported and to date it is impossible to predict whether a protein will be imported into an organelle with a given transit peptide. As such, it is advantageous to have a diversity of potential transit peptides from which the most efficient candidate can be chosen to target a protein of interest to an organelle. A number of plant transit peptides are known which direct mature proteins to mitochondria or chloroplast organells. These transit peptides are diverse in structure (length and amino acid sequence) and there is no strong consensus sequence identifying them. In addition, there is no obvious clear relationship between chloroplast targeting and mitochondrial targeting transit sequences. This invention describes a number of chloroplast-targeting and mitochondria-targeting transit peptides for (maize) aminoacyl-tRNA synthetases. These sequences will find utility in directing both aminoacyl-tRNA synthetase and other proteins into these organelles.
Accordingly, the availability of nucleic acid sequences encoding all or a portion of these enzymes would facilitate studies to better understand protein synthesis in plants, provide genetic tools for the manipulation of gene expression, protein targeting to specific organells and provide possible targets for herbicides.
SUMMARY OF THE INVENTION
The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a first polypeptide of at least 116 amino acids that has at least 85% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a rice seryl-tRNA synthetase polypeptide of SEQ ID NO:16, a soybean seryl-tRNA synthetase polypeptide of SEQ ID NO:18, or a wheat seryl-tRNA synthetase polypeptide of SEQ ID NO:20. The present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above.
The present invention also relates to isolated polynucleotides comprising a nucleotide sequence encoding a second polypeptide of at least 483 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a corn threonyl-tRNA synthetase polypeptide of SEQ ID NO:22, a rice threonyl-tRNA synthetase polypeptide of SEQ ID NO:24, a soybean threonyl-tRNA synthetase polypeptide of SEQ ID NO:26, or a wheat threonyl-tRNA synthetase polypeptide of SEQ ID NO:28. The present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above.
The present invention relates to an isolated polynucleotide comprising a nucleotide sequence encoding a first polypeptide of at least 30 amino acids that has at least 70% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12 and 14.
It is preferred that the isolated polynucleotides of the claimed invention consists of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 and 27 that codes for the polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and 28. The present invention also relates to an isolated polynucleotide comprising a nucleotide sequences of at least one of 40 (preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 and 27 and the complement of such nucleotide sequences.
The present invention relates to a chimeric gene comprising an isolated polynucleotide of the present invention operably linked to suitable regulatory sequences.
The present invention also relates to the identification of transit peptides associated with aminoacyl-tRNA synthetases of the instant invention and the use of those transit peptides to target aminoacyl-tRNA synthetases and other operably linked proteins to specific organelles within plant cells. Transit peptide amino acid sequences are located just upstream of the mature aminoacyl-tRNA synthetase polypeptide sequences disclosed in the instant invention.
The present invention relates to an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention. The host cell may be eukaryotic, such as a yeast or a plant cell, or prokaryotic, such as a bacterial cell. The present invention also relates to a virus, preferably a baculovirus, comprising an isolated polynucleot
Famodu Omolayo O.
Simmons Carl
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