Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Plant proteins – e.g. – derived from legumes – algae or...
Reexamination Certificate
1999-09-03
2001-10-02
Low, Christoper S. F. (Department: 1653)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Plant proteins, e.g., derived from legumes, algae or...
C530S350000, C530S300000, C530S324000, C530S370000, C435S069100
Reexamination Certificate
active
06297360
ABSTRACT:
BACKGROUND OF THE INVENTION
The hypersensitive response (HR) of higher plants is characterized by the rapid, localized death of plant cells at the site of pathogen invasion. HR occurs during incompatible pathogen/host interactions, such as when a microorganism that normally causes a disease in its host plant infects a non-host plant. The response is associated with resistance against a variety of pathogens, including nematodes, fungi, viruses, and bacteria. For a review of the hypersensitive response, see Dixon et al., Annu Rev Phytopathol 32:479 (1994) and Godiard et al., Curr Opin Genet Dev 4:662 (1994).
The ability of phytopathogenic bacteria to cause HR in resistant or non-host plants is controlled by a cluster of highly conserved bacterial genes named hypersensitive response and pathogenicity (hrp) genes. Most hrp genes are involved in forming a protein secretion apparatus for harpins, heat-stable and proteinaceous proteins which elicit HR when infiltrated into the leaf intercellular spaces of non-host plants. It is known that, when added to a plant cell culture, harpins induce the exchange of H
+
and K
+
across the plasmalemma to generate active oxygen species (Baker et al., Plant Physiol 102:1341 [1993]).
SUMMMARY OF THE INVENTION
This invention relates to an isolated nucleic acid encoding an amphipathic protein-1 (AP-1) which decreases the extent or duration of HR in a plant. The term “amphipathic protein-1” refers to any natural or man-made variant of AP-1. For example, the nucleic acid can have the sequence of SEQ ID NO:1 (shown below), have a sequence which hybridizes under stringent conditions to SEQ ID NO:1, or have a sequence which encodes SEQ ID NO:2.
A vector and transformed host cell containing such a nucleic acid is included within the scope of this invention. A vector is any nucleic acid molecule or virus containing regulatory elements or reporter genes for the purpose of, but not limited to, expression in prokaryotic or eukaryotic cells or organisms. A transformed host cell is a host cell into which (or into an ancestor of which) has been introduced, by means of molecular biological techniques, a nucleic acid encoding an AP-1 of this invention. After introduction into the cell, this nucleic acid can exist extrachromosomally or become integrated into the host genome.
The present invention also relates to a substantially pure AP-1, such as SEQ ID NO:2 (shown below) or a protein which differs from SEQ ID NO:2 by at least one conservative amino acid substitution.
A “nucleic acid” encompasses both RNA and DNA, including cDNA, genomic DNA, and synthetic (e.g., chemically synthesized or modified) DNA. The nucleic acid may be double-stranded or single-stranded. Where single stranded, the nucleic acid may be a sense strand or an antisense strand. An “isolated nucleic acid” refers to a nucleic acid which may be flanked by non-natural sequences, such as those of a plasmid or virus. Thus, the nucleic acid can include none, some, or all of the 5′ non-coding (e.g., promoter) sequences which are immediately contiguous to the coding sequence. The term, therefore, includes, for example, a recombinant DNA which is incorporated into a vector including an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences. The term also includes a recombinant DNA or RNA which is part of a hybrid gene encoding an additional polypeptide sequence. Moreover, the term is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state.
By “hybridizes under stringent conditions” is meant the conditions in which a nucleic acid forms a stable, sequence-specific, non-covalent bond with the nucleic acid of SEQ ID NO:1 in solution or on solid support under the low salt and high temperature conditions regarded as stringent and set forth in Sambrook et al (Molecular Cloning, A Laboratory Manual, Sambrook, J., Fritsch, E. F., and Maniatis, T., 2nd ed. [1989] Cold Spring Harbor Laboratory Press). For example, reference nucleic acids such as SEQ ID NO:1 can be immobilized on nitrocellulose filters. Any nucleic acids specifically and non-covalently binding to the immobilized reference nucleic acids in the presence of 0.2×SSC (1.75 g/l NaCl, 0.88 g/l Na
3
citrate.2H
2
O; pH 7.0) and 0.1% (w/v) sodium dodecylsulfate at 68° C. are considered to be hybridized under stringent conditions.
The term “substantially pure” as used herein in reference to a given AP-1 polypeptide means that the polypeptide is substantially free from other compounds, such as those in cellular material, viral material, or culture medium, with which the polypeptide may have been associated (e.g., in the course of production by recombinant DNA techniques or before purification from a natural biological source). Polypeptides are substantially free from other compounds when they are within preparations that are at least 60% by weight (dry weight) the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
Where a particular polypeptide or nucleic acid molecule is said to have a specific percent identity to a reference polypeptide or nucleic acid molecule of a defined length, the percent identity is relative to the reference polypeptide or nucleic acid molecule. Thus, a polypeptide that is 50% identical to a reference polypeptide that is 100 amino acids long can be a 50 amino acid polypeptide that is completely identical to a 50 amino acid long portion of the reference polypeptide. It might also be a 100 amino acid long polypeptide which is 50% identical to the reference polypeptide over its entire length.
For polypeptides, the length of the reference polypeptide sequence will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids, 50 amino acids, or 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably at least 100 nucleotides (e.g., 150, 200, 250, or 300 nucleotides).
In the case of polypeptide sequences that are less than 100% identical to a reference sequence, the non-identical positions are preferably, but not necessarily, conservative substitutions for the reference sequence. Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine. For example, a 10 amino acid polypeptide is said to be at least 80% conserved if it differs from a reference polypeptide by no more than two non-conservative substitutions. An AP-1 polypeptide of this invention is preferably 70% conserved, more preferably 80% conserved, and most preferably 90% conserved as compared to SEQ ID NO:2.
Sequence identity can be measured using sequence analysis software, e.g., the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705 with the default parameters as specified therein.
The BLAST programs, provided as a service by the National Center for Biotechnology Information, are useful for making sequence comparisons. The programs are described in detail by Karlin et al., Proc Natl Acad Sci USA 87:2264 (1990) and 90:5873 (1993), and Altschul et al., Nucl Acids Res 25:3389 (1997) and are available on the Internet.
The isolation and ch
Feng Teng-yung
Lin Hae-jan
Academia Sinica
Bugaisky Gabriele E.
Fish & Richardson P.C.
Low Christoper S. F.
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