Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or...
Reexamination Certificate
1998-07-17
2003-11-04
Smith, Lynette R. F. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
C435S069100, C435S070100, C435S410000, C435S419000, C536S023100, C536S023600, C536S023200, C800S295000, C800S306000, C800S298000
Reexamination Certificate
active
06642434
ABSTRACT:
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
Not applicable.
BACKGROUND OF THE INVENTION
Vitamin E is an essential component of mammalian diets. Epidemiological evidence indicates that Vitamin E supplementation results in decreased risk for cardiovascular disease and cancer, aids in immune function, and generally prevents or slows a number of degenerative disease processes in humans (Traber and Sies,
Annu. Rev. Nutr
. 16:321-347, 1996). Vitamin E functions in stabilizing the lipid bilayer of biological membranes (Skrypin and Kagan,
Biochim. Biophys. Acta
815:209 1995; Kagan,
N.Y. Acad. Sci
. p 121, 1989; Gomez-Fernandez et al.,
Ann. N.Y. Acad. Sci
. p 109, 1989), reducing polyunsaturated fatty acid (PUFA) free radicals generated by lipid oxidation (Fukuzawa et al.,
Lipids
17: 511-513, 1982), and quenching singlet oxygen species (Fryer,
Plant Cell Environ
. 15(4):381-392, 1992).
Vitamin E, or &agr;-tocopherol, belongs to a class of lipid-soluble antioxidants that includes &agr;, &bgr;, &ggr;, and &dgr;-tocopherols and &agr;, &bgr;, &ggr;, and &dgr;-tocotrienols. Although &agr;, &bgr;, &ggr;, and &dgr;-tocopherols and &agr;, &bgr;, &ggr;, and &dgr;-tocotrienols are sometimes referred to collectively as “Vitamin E” in the popular press, Vitamin E is properly defined chemically solely as &agr;-tocopherol. Of the various tocopherols present in foodstuff, &agr;-tocopherol is the most significant for human health both because it is the most bioactive of the tocopherols and also because it is the tocopherol most readily absorbed and retained by the body (Traber and Sies,
Annu. Rev. Nutr
. 16:321-347, 1996). The in vivo antioxidant activity of &agr;-tocopherol is higher than the antioxidant activities of &bgr;, &ggr;, and &dgr;-tocopherol (Kamal-Eldin and Appelqzvist
Lipids
31:671-701, 1996).
Only plants and certain other photosynthetic organisms, including cyanobacteria, synthesize tocopherols. Therefore, dietary tocopherols are obtained almost exclusively from plants. Plant tissues vary considerably in total tocopherol content and tocopherol composition. The predominant tocopherol in green, photosynthetic plant tissues often is &agr;-tocopherol. Leaf tissue can contain from 10-50 &mgr;g total tocopherols/gram fresh weight.
Non-green plant tissues and organs exhibit a wider range of both total tocopherol levels and tocopherol compositions. In general, most of the major food staple corps (e.g., rice, corn, wheat, potato) produce low to extremely low levels of total tocopherols, of which only a small percentage is &agr;-tocopherol (Hess, Vitamin E, &agr;-tocopherol, In Antioxidants in Higher Plants, R. Alscher and J. Hess, Eds. 1993, CRC Press, Boca Raton. pp 111-134). Oil seed crops generally contain much higher levels of total tocopherols; however, &agr;-tocopherol is present only as a minor component and &bgr;, &ggr;, and &dgr;-tocopherols and tocotrienols predominate (Taylor and Barnes,
Chemy Ind
., October:722-726, 1981).
Daily dietary intake of 15-30 mg of vitamin E is recommended to obtain optimal plasma &agr;-tocopherol levels. It is quite difficult to achieve this level of vitamin E intake from the average American diet. For example, one could obtain the recommended daily dose of Vitamin E by daily consumption of over 750 grams of spinach leaves (in which &agr;-tocopherol comprises 60% of total tocopherols) or 200-400 grams of soybean oil.
One alternative to relying on diet alone to obtain the recommended levels of vitamin E is to take a vitamin E supplement. However, most vitamin E supplements are synthetic vitamin E having six stereoisomers, whereas natural vitamin E vitamin is a single isomer. Furthermore, supplements tend to be relatively expensive, and the general population is disinclined to take vitamin supplements on a regular basis.
Although tocopherol function in plants has been less extensively studied than tocopherol function in mammalian systems, it is likely that the analogous functions performed by tocopherols in animals also occur in plants. In general, plant tocopherol levels have been found to increase with increases in various stresses, especially oxidative stress. Increased &agr;-tocopherol levels in crops are associated with enhanced stability and extended shelf life of fresh and processed plant products (Peterson,
Cereal
-
Chem
72(1):21-24, 1995; Ball,
Fat
-
soluble vitamin assays in food analysis. A comprehensive review
. London: Elsevier Science Publishers LTD, 1988).
Vitamin E supplementation of swine, beef, and poultry feeds has been shown to significantly increase meat quality and extend the shelf life of post-processed meat products by retarding post-processing lipid oxidation, which contributes to the formation of undesirable flavor components (Ball, supra 1988; Sante and Lacourt,
J. Sci. Food Agric
. 65(4):503-507, 1994; Buckley et al.,
J. of Animal Science
73:3122-3130, 1995).
What would be useful for the art is a method to increase the ratio of &agr;-tocopherol to &ggr;-tocopherol in seeds, oils, and leaves from crop and forage plants, or a method for producing natural vitamin E in nonphotosynthetic bacteria or fungi using a large scale fermentation process. Increasing &agr;-tocopherol levels in crop plants would increase the amount of &agr;-tocopherol obtained in the human diet, and would enhance the stability and shelf life of plants and plant products. The meat industry would benefit from the development of forage plants having increased levels of vitamin E.
BRIEF SUMMARY OF THE INVENTION
The present invention is based on an isolated DNA fragment including a coding sequence for a &ggr;-tocopherol methyltransferase.
The invention is also a heterologous genetic construct comprising a &ggr;-tocopherol methyltransferase coding sequence operably connected to a plant, bacterial, or fungal promoter not natively associated with the &ggr;-tocopherol methyltransferase coding sequence.
Another aspect of the present invention is a method of altering the tocopherol profile of a plant comprising the steps of: (a) providing a heterologous genetic construct comprising a &ggr;-tocopherol methyltransferase coding sequence operably connected to a plant promoter not natively associated with the coding sequence; and (b) introducing the construct into the genome of a plant.
The present invention is also directed toward transgenic plants which have an altered ratio of &agr;-tocopherol to &ggr;-tocopherol, thus increasing the nutritive value of the plants and products therefrom for human and animals.
In another embodiment, the invention is a plant comprising in its genome a heterologous genetic construct comprising a &ggr;-tocopherol methyltransferase coding sequence operably connected to a promoter that is functional in plants.
It is an object of the present invention to provide a genetic construct comprising a coding sequence for a &ggr;-tocopherol methyltransferase operably connected to a plant promoter not natively associated with the coding sequence which when expressed in a plant comprising the construct in its genome results in an alteration in the ratio of &agr;-tocopherol: &ggr;-tocopherol in the plant, relative to an untransformed wild-type plant.
It is an object of this invention to provide a plant having an altered &agr;-tocopherol: &ggr;-tocopherol ratio.
Other objects, features, and advantages of the invention will become apparent upon review of the specification and claims.
REFERENCES:
Alscher et al. Anitoxidants in Higher Plants. CRC press. 1993.*
Smith at al. Nature. 1988. Vol. 334: 724-726, 1988.*
Kaneko et al. 1995. DNA Research. 2: 153-166 (Accession number enclosed: BAA10562), 1995.*
Fillatti et al. Biotechnology. 1987. Vol. 5: 726-730, 1987.*
d'Harlingue, Alain and Bilal Camara, “Plastid Enzymes of Terpenoid Biosynthesis; Purification and Characterization of &ggr;-Tocopherol Methyltransferase fromCapsicumChromoplasts”,J. Biol. Chem.260(28): 15200-15203 (1985).
Hess, John L., “Vitamin E, &agr;-Tocopherol” inAntioxidants in Highter Plantsedited by Alscher, Ruth G. and John L. Hess, CRC Press, Inc. pp. 1
DellaPenna Dean
Shintani David K.
Quarles & Brady LLP
Smith Lynette R. F.
University of Community College System of Nevada
Zaghmout Ousama
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