Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or...
Reexamination Certificate
2000-05-22
2003-02-25
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
C800S276000, C800S287000, C800S288000, C800S282000, C800S291000, C800S294000, C800S298000, C800S306000, C435S320100, C435S419000, C435S004000, C435S008000, C435S468000, C435S469000, C435S441000
Reexamination Certificate
active
06525245
ABSTRACT:
FIELD OF THE INVENTION
The current invention is directed toward a method for identifying components involved in signal transduction pathways in higher plants. In particular, the present invention relates to a method to elucidate key nucleic acid sequences of components involved in the signal transduction pathway that communicates mitochondrial function and metabolic status to nuclear gene expression. The present invention also relates to a method for identifying key nucleic acid sequences of components in the signal transduction pathway between branched chain amino acid biosynthetic pathways and nuclear gene expression. Additionally, the present invention also relates to a polynucleotide that encodes a portion of an AOX promoter, AOX1a, operably linked to a luciferase reporter gene. The present invention also relates to a recombinant vector, transformed cells, and transformed organisms containing this polynucleotide.
BACKGROUND OF THE INVENTION
Mitochondria play a crucial role in the overall physiology of an organism and are a primary site of energy production in all eukaryotic cells. The products of carbohydrate, lipid, and protein catabolism enter the mitochondria and are oxidized by the tricarboxylic acid cycle (TCA) leading to the production of reducing equivalents (NADH and succinate), ATP, and CO
2
(Mackenzie et al., (1999) Plant Cell 11:571-586). Energy production occurs as the reducing equivalents NADH and succinate are oxidized and the electrons are fed into a series of enzyme complexes called the mitochondrial electron transport (or respiratory) chain (mtETC). In most eukaryotes, the electrons are passed through the mtETC via the cytochrome respiratory pathway (FIG.
1
). In this pathway, electrons move through the mtETC from complex I (NADH dehydrogenase), or complex II (succinate dehydrogenase) or the internal rotenone-insensitive, external and outer membrane NAD(P)H dehydrogenases to the ubiquinone pool. Electrons are then transferred to complex III, then to diffusible cytochrome c and finally to complex IV, which utilizes the electrons to reduce oxygen to water. At several points in the pathway, protons are pumped from the matrix to the intermembrane space, establishing a proton gradient across the inner membrane. The proton gradient is then utilized by complex V (ATPase) to drive the synthesis of ATP. Hence, the cytochrome pathway couples oxidation to the synthesis of ATP. ATP is primary source of chemical energy in the cell.
In higher plants, some fungi, unicellular green algae, and trypanosomes, however, an alternative mtETC pathway exists (referred to as the alternative respiratory pathway) (FIG.
1
). In the alternative respiratory pathway, electrons can move from the ubiquione pool to alternative oxidase (AOX), which also reduces oxygen to water (Mackenzie et al., (1999) Plant Cell 11:571-586). AOX does not pump protons and therefore, this pathway results in either a much lower or no establishment of a proton gradient. The end result of the alternative oxidase pathway is an uncoupling of electron transport from ATP synthesis wherein the energy from electron transport is dissipated as heat instead of being harnessed for the production of ATP. The function of the alternative pathway has yet to be fully elucidated, however, proposed functions include 1) an overflow for electrons when the cytochrome pathway is saturated; 2) a means of allowing continued carbon skeleton turnover and conversion when cellular energy is high; and 3) an elimination system for reactive oxygen species. In addition, AOX activity has been shown to be influenced by environmental, developmental, chemical and tissue specific signals (Aubert et al., (1997) Plant J. 11:649-657; and Mackenzie et al., (1999) Plant Cell 11:571-586).
In order to preserve mitochondrial integrity, plants must perceive and respond to numerous developmental changes and environmental stresses. It is of particular importance for plants, that must survive in place, to be able to adapt to harsh environmental conditions. At the molecular level, one mechanism plants and other organisms have in their repertoire to cope with such conditions is the alteration of protein expression. For example, plants alter the expression of proteins in the mtETC as a means to ensure that electron flow is correctly partitioned between the cytochrome and alternative oxidase pathways to meet the energy demands of the cell at any given time. Additionally, numerous environmental stresses and developmental signals can alter the protein profile of mitochondria (Sachs et al., (1986) Ann. Rev. of Plant Physiol. 37:363-376). For example, in response to heat-stress a new class of proteins is induced (the so called “heat shock” proteins) to help ameliorate the impact of heat-stress on the plant (Waters et al., (1996) J. Exp. Bot. 47:325-338). Plants also alter protein expression in response to a number of other environmental stresses including but not limited to: phosphate deficiency, cold stress, aging, salt stress and elevated CO
2
levels.
The means by which plant mitochondria alter protein expression is complicated and remains largely enigmatic. The mitochondria has a genome of its own, however, only 10% of the genes needed by the mitochondria are encoded by its genome (Schuster et al., (1994) Ann. Rev. Plant Mol. Biol. 45:61-78). Thus, most mitochondrial proteins are the products of nuclear genes that are imported into the mitochondria from the cytosol following their synthesis. The means by which mitochondrial status is communicated to the nucleus is through a signal transduction pathway (de Winde et al., (1993)
Saccharomyces cerevisiae
. Prog. Nucleic Acid Res. Mol. Biol. 46:51-91; Poyton et al., (1 996) Annu. Rev. Biochem. 65:563-607; and Zitomer et al., (1992)
Saccharomyces cerevisiae
. Microbiol. Rev. 56:1-11). Signal transduction pathways are one mechanism that the cell uses to respond to the surrounding environment (Lewin B. (1997) Genes VI, Oxford University Press, 1053-1082). Through a series of reactions involving numerous protein components and secondary messengers, signal transduction pathways communicate environmental status to the nucleus so that gene expression is tailored to meet the protein demands resulting from the environmental or developmental changes.
Organisms must also regulate the expression of genes encoding proteins involved in branched chain amino acid biosynthesis and likely accomplish this through signal transduction pathways. Acetolactate synthase catalyzes the first committed step in the pathway and its inhibition leads to metabolic perturbation, which results in a lack of branched chain amino acids (Aubert et al., (1997) Plant J. 11:649-657; and Bryan J. K. (1980) The Biochemistry of Plants: A Comprehensive Treatise, Academic Press 5:403-452). Inhibition of branched chain amino acid biosynthesis has been shown to result in an accumulation of AOX protein and transcript (Aubert et al., (1997) Plant J. 11:649-657). Specifically, the herbicides sulfmometuron methyl, chlorsulfuron and sceptor have all been shown to inhibit branched chain amino acid biosynthesis and result in an increase in AOX transcription (Aubert et al., (1997) Plant J. 11:649-657). Hence, characterizing the signal transduction pathway between branched chain amino acid biosynthetic pathways and nuclear gene expression is of particular interest in plants because of the impact that herbicides can have on overall plant metabolism. For example, understanding the mechanism by which specific metabolic enzyme expression is altered in response to herbicide application may provide a means to control the overall response of plants to herbicides.
Research methods to efficiently and comprehensively determine the mechanism of signal transduction pathways have not been fully developed. Characterizing key components involved in signal transduction pathways is a formidable challenge due to the number of protein components that participate in transmitting the signal, the complex biochemical mechanism by which the signal is transmitted, and the impact of signal on
Fox David T.
Kubelik Anne
The Board of Regents of the University of Nebraska
LandOfFree
Method for identifying components involved in signal... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method for identifying components involved in signal..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for identifying components involved in signal... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3139878