Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Reexamination Certificate
1999-11-04
2002-03-12
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
C435S252300, C435S254100, C435S255100, C435S320100, C536S023200, C536S023400, C536S023700
Reexamination Certificate
active
06355462
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 berberine-bridge-forming enzyme in plants and seeds.
BACKGROUND OF THE INVENTION
Pathogens annually cause billions of dollars in damage to crops worldwide. Consequently, an increasing amount of research has been dedicated to developing novel methods for controlling plant diseases. Such studies have centered on the plant's innate ability to resist pathogen invasion in an effort to support the plant's own defenses to counter pathogen attacks (Staskawicz et al. (1995)
Science
268:661-667; Baker et al. (1997)
Science
276:726-733). One such defense mechanism under study is known as systemic acquired resistance (SAR; reviewed in Ryals et al. (1996)
Plant Cell
8:1809-1819). SAR is defined as a generalized defense response, which is often induced by avirulent pathogens and provides enhanced resistance to a broad spectrum of virulent pathogens. Avirulent pathogens carry an avirulence (avr) gene whose product can be recognized by the product of a corresponding resistance (R) gene carried by plants. Such recognition triggers both a programmed cell death response, known as the hypersensitive response (HR), around the point of pathogen infection and release of a systemic SAR-inducing signal (Hammond-Kosack and Jones (1996)
Plant Cell
8:1773-1791). After a rapid, localized HR, the elevated state of resistance associated with SAR is effective throughout the plant for a period of time ranging from several days to a few weeks. Coinciding with the onset of SAR is the transcriptional activation of the pathogenesis-related (PR) genes. These genes encode proteins that exhibit antimicrobial activities (Ward et al. (1991)
Plant Cell
3:1085-1094).
The berberine bridge-forming enzyme ((S)-reticuline:oxygen oxidoreductase (methylene-bridge-forming); EC 1.5.3.9) is a vesicular plant enzyme that catalyzes the formation of the berberine bridgehead carbon of (S)-scoulerine from the N-methyl carbon of (S)-reticuline in a specific, unparalleled reaction along the biosynthetic pathway that leads to benzophenanthridine alkaloids. Cytotoxic benzophenanthridine alkaloids are accumulated in certain species of Papaveraceae and Fumariaceae in response to pathogenic attack and, therefore, function as phytoalexins. The berberine bridge enzyme has been purified to homogeneity from elicited cell-suspension cultures of
Eschscholtzia californica
. The mature protein has a molecular weight of 57,352, excluding carbohydrate. The berberine bridge enzyme was heterologously expressed in a catalytically active form in
Saccharomyces cerevisiae
. Southern hybridization with genomic DNA suggests that there is only one gene for the enzyme in the
E. californica
genome. Hybridized RNA blots from elicited
E. californica
cell-suspension cultures reveals a rapid and transient increase in poly(A)+RNA levels that preceded both the increase in enzyme activity and the accumulation of benzophenanthridine alkaloids, emphasizing the integral role of the berberine bridge enzyme in the plant response to pathogens (Dittrich and Kutchan (1991)
Proc Natl Acad Sci USA
88:9969-9973).
SUMMARY OF THE INVENTION
The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a first polypeptide of at least 25 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 a corn berberine-bridge-forming enzyme polypeptide of SEQ ID NO:2, a rice berberine-bridge-forming enzyme polypeptide of SEQ ID NO:4, a soybean berberine-bridge-forming enzyme polypeptide of SEQ ID NO:6, and a wheat berberine-bridge-forming enzyme polypeptide of SEQ ID NO:8. The present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above.
It is preferred that the isolated polynucleotide of the claimed invention consists of a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, and 7 that codes for the polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, and 8. The present invention also relates to an isolated polynucleotide comprising a nucleotide sequences of at least 30 (preferably at least 40) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, and 7 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 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 polynucleotide of the present invention or a chimeric gene of the present invention.
The present invention relates to a process for producing an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention, the process comprising either transforming or transfecting an isolated compatible host cell with a chimeric gene or isolated polynucleotide of the present invention.
The present invention relates to a berberine-bridge-forming enzyme polypeptide of at least 25 amino acids comprising at least 70% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, and 8.
The present invention relates to a method of selecting an isolated polynucleotide that affects the level of expression of a berberine-bridge-forming enzyme polypeptide in a host cell, preferably a plant cell, the method comprising the steps of:
constructing an isolated polynucleotide of the present invention or an isolated chimeric gene of the present invention;
introducing the isolated polynucleotide or the isolated chimeric gene into a host cell;
measuring the level a berberine-bridge-forming enzyme polypeptide in the host cell containing the isolated polynucleotide; and
comparing the level of a berberine-bridge-forming enzyme polypeptide in the plant cell containing the isolated polynucleotide with the level of a berberine-bridge-forming enzyme polypeptide in a plant cell that does not contain the isolated polynucleotide.
The present invention relates to a method of obtaining a nucleic acid fragment encoding a substantial portion of a berberine-bridge-forming enzyme polypeptide gene, preferably a plant berberine-bridge-forming enzyme polypeptide gene, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least 30 (preferably at least) 40 contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer. The amplified nucleic acid fragment preferably will encode a portion of a berberine-bridge-forming enzyme amino acid sequence.
The present invention also relates to a method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a berberine-bridge-forming enzyme polypeptide comprising the steps of: probing a cDNA or genomic library with an isolated polynucleotide of the present invention; identifying a DNA clone that hybridizes with an isolated polynucleotide of the present invention; isolating the identified DNA clone; and sequencing the cDNA or genomic fragment that comprises the isolated DNA clone.
The present invention relates to a composition comprising an isolated polynucleotide of the present invention.
The present invention relates to an isolated polynucl
Falco Saverio Carl
Famodu Omolayo O.
Han Feng
Rafalski J. Antoni
E.I. du Pont de Nemours and Company
Prouty Rebecca E.
Rao Manjunath N.
LandOfFree
Disease resistance factor does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Disease resistance factor, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Disease resistance factor will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2844406