Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
2000-03-29
2004-08-31
Fredman, Jeffrey (Department: 1634)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C800S295000, C800S320300, C800S278000, C800S287000
Reexamination Certificate
active
06784346
ABSTRACT:
BACKGROUND OF THE INVENTION
Thioredoxins are small (about 12 kDa) thermostable proteins with catalytically active disulfide groups. This class of proteins has been found in virtually all organisms, and has been implicated in myriad biochemical pathways (Buchanan et al., 1994). The active site of thioredoxin has two redox-active cysteine residues in a highly conserved amino acid sequence; when oxidized, these cysteines form a disulfide bridge (—S—S—) that can be reduced to the sulfhydryl (—SH) level through a variety of specific reactions. In physiological systems, this reduction may be accomplished by reduced ferredoxin, NADPH, or other associated thioredoxin-reducing agents. The reduced form of thioredoxin is an excellent catalyst for the reduction of even the most intractable disulfide bonds.
Generally only one kind of thioredoxin is found in bacterial or animal cells. In contrast, photosynthetic organisms have three distinct types of thioredoxin. Chloroplasts contain a ferredoxin/thioredoxin system comprised of ferredoxin, ferredoxin-thioredoxin reductase and thioredoxins f and m, which function in the light regulation of photosynthetic enzymes (Buchanan, 1991; Scheibe, 1991). The other thioredoxin enzyme system is analogous to that established for animals and most microorganisms, in which thioredoxin (h-type in plants) is reduced by NADPH and NADPH-thioredoxin reductase (NTR) (Johnson et al., 1987a: Florencio et al., 1988; Suske et al., 1979). The reduction of thioredoxin h by this system can be illustrated by the following equation:
Thioredoxin is a component of two types of enzyme systems in plants. Chloroplasts contain a ferredoxin/thioredoxin system comprised of ferredoxin, ferredoxin-thioredoxin reductase and thioredoxins f and m, that are involved in the light regulation of photosynthetic enzymes (Buchanan, 1991; Scheibe, 1991). The other enzyme system, the NADP-thioredoxin system or NTS, is analogous to the system established for animals and most microorganisms, in which thioredoxin (h-type in plants) is reduced by NADPH and NADPH-thioredoxin reductase (NTR) (Johnson et al., 1987a; Florencio et al., 1988; Suske et al., 1979). Thioredoxin h is widely distributed in plant tissues and exists in mitochondria, endoplasmic reticulum (ER) and cytosol (Bodenstein-Lang et al., 1989; Marcus et al., 1991).
Plant thioredoxin h is involved in a wide variety of biological functions. The presence of multiple forms of thioredexoin h protein has also been reported in plant seeds (Bestermann et al., 1983). In wheat, three different thioredoxin have been characterized (Vogt and Follman, 1986). Thioredoxin h functions in the reduction of intramolecular disulfide bridges of a variety of low molecular-weight, cystine-rich proteins, including thionins (Johnson et al., 1987b), protease inhibitors and chloroform/methanol-soluble proteins (CM proteins or alpha-amylase inhibitors) (Kobrehel et al., 1991). It is likely that cytoplasmic thioredoxins participate in developmental processes: for example thioredoxin h has been shown to function as a signal to enhance metabolic processes during germination and seedling development (Kobrehel et al., 1992; Lozano et al., 1996; Besse et al., 1996): Thioredoxin h has also been demonstrated to be involved in self-incompatibility in
Phalaris coerulescens
(Li et al., 1995) and
Brassica napus
(Bower et al., 1996). Several functions have been hypothesized for rice thioredoxin h, which is believed to be involved in translocation in sieve tubes (Ishiwatari et al., 1995).
The NTS has been shown to improve dough quality. The improvement in dough strength and bread quality properties of poor-quality wheat flour resulting from the addition of thioredoxin (Wong et al., 1993; Kobrehel et al., 1994) may be attributable to the thioredoxin-catalyzed reduction of intramolecular disulfide bonds in the flour proteins, specifically the glutenins, resulting in the formation of new intermolecular disulfide bonds (Besse and Buchanan, 1997). Thus, the addition of exogenous thioredoxin promotes the formation of a protein network that produces flour with enhanced baking quality. Kobrehel et al., (1994) have observed that the addition of thioredoxin h to flour of non-glutenous cereals such as rice, maize and sorghum promotes the formation of a dough-like product. Hence, the addition of exogenous thioredoxin may be used to produce baking dough from non-glutenous cereals.
In addition, it has been shown that reduction of disulfide protein allergens in wheat and milk by thioredoxin decreases their allergenicity (Buchanan et al., 1997; del Val et al., 1999). Thioredoxin treatment also increases the digestibility of the major allergen of milk (&bgr;-lactoglobulin) (del Val et al., 1999), as well as other disulfide proteins (Lozano et al., 1994; Jiao et al., 1992). Therefore, the manipulation of the NTS offers considerable promise for production of nutraceutical and pharmaceutical products. A more detailed discussion of the benefits of adding exogenous thioredoxin to food products is presented in U.S. Pat. No. 5,792,506 to Buchanan et at.
cDNA clones encoding thioredoxin h have been isolated from a number of plant species, including
Arabidopsis thaliana
(Rivera-Madrid et al., 1993; Rivera-Madrid et al., 1995),
Nicotiana tabacum
(Marty and Meyer, 1991; Brugidou et al., 1993),
Oryza sativa
(Ishiwatari et al., 1995),
Brassica napus
(Bower et al., 1996),
Glycine max
(Shi and Bhattacharyya, 1996), and
Triticum aestivum
(Gautier et al., 1998). More recently, two cDNA clones encoding wheat thioredoxin h have been isolated and characterized (Gautier et al., 1998). The
Escherichia coli
NTR gene has been first isolated (Russel and Model, 1988) and the three-dimensional structure of the protein has been analyzed (Kuriyan et al., 1991). Some other NTR genes have been isolated and sequenced from bacteria, fungi and mammals. Recently, Jacquot et al., (1994) have reported a successful isolation and sequencing of two cDNAs encoding the plant
A. thaliana
NTRs. The subsequent expression of the recombinant
A. thaliana
NTR protein in
E. coli
cells (Jacquot et al., 1994) and its first eukaryotic structure (Dai et al., 1996) have also been reported.
Here we disclose value-added traits in transgenic grains, such as barley (Cho et al., 1999b)., wheat, and sorghum, overexpressing thioredoxin
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Cho, M.-J., et al., “Subcellular Targeting of Barley Hordein Promoter-uidA Fusions in Transgenic Barley Seed”In Vitro Cellular and Developmental Biology 34(3)part 2:48A (1998) abstract only.
Buchanan Bob B.
Cho Myeong-Je
Lemaux Peggy G.
Marx Corina
Wong Joshua
Fredman Jeffrey
Morrison & Foerster / LLP
Switzer Juliet C.
The Regents of the University of California
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