Amino polyol amine oxidase polynucleotides and related...

Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide confers pathogen or pest resistance

Reexamination Certificate

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C800S278000, C800S320100, C800S320200, C800S320300, C800S317400, C800S314000, C800S312000, C800S322000, C800S306000, C800S320000, C800S288000, C800S286000, C435S468000, C435S419000, C435S183000, C435S195000, C435S196000, C435S320100, C435S069100, C435S070100, C435S071100, C435S348000, C536S023200, C536S024100, C536S023700

Reexamination Certificate

active

06211434

ABSTRACT:

TECHNICAL FIELD
The present invention relates generally to the detection and isolation of fumonisin and AP1 degrading enzymes and to compositions and methods for the in vivo detoxification or degradation of fumonisin or its hydrolysis product AP1. This method has broad application in agricultural biotechnology and crop agriculture and in the improvement of food grain quality.
BACKGROUND OF THE INVENTION
Fungal diseases are common problems in crop agriculture. Many strides have been made against plant diseases as exemplified by the use of hybrid plants, pesticides and improved agricultural practices. However, as any grower or home gardener can attest, the problems of fungal plant disease continue to cause difficulties in plant cultivation. Thus, there is a continuing need for new methods and materials for solving the problems caused by fungal diseases of plants.
These problems can be met through a variety of approaches. For example, the infectious organisms can be controlled through the use of agents that are selectively biocidal for the pathogens. Another method is interference with the mechanism by which the pathogen invades the host crop plant. Yet another method, in the case of pathogens that cause crop losses, is interference with the mechanism by which the pathogen causes injury to the host crop plant. Still another method, in the case of pathogens that produce toxins that are undesirable to mammals or other animals that feed on the crop plants, is interference with toxin production, storage, or activity. This invention falls into the latter two categories.
Since their discovery and structural elucidation in 1988 (Bezuidenhout et al., Journal
Chem Soc, Chem Commun
1988: 743-745 (1988)), fumonisins have been recognized as a potentially serious problem in maize-fed livestock. They are linked to several animal toxicoses including leukoencephalomalacia (Marasas, et al.,
Onderstepoort Journal of Veterinary Research
55: 197-204 (1988); Wilson, et al.,
American Association of Veterinary Laboratory Diagnosticians: Abstracts
33
rd Annual Meeting
, Denver, Colo., Oct. 7-9, 1990, Madison, Wis., USA) and porcine pulmonary edema (Colvin, et al.,
Mycopathologia
117: 79-82 (1992)). Fumonisins are also suspected carcinogens (Geary W (1971)
Coord Chem Rev
7: 81; Gelderblom, et al.,
Carcinogenesis
12: 1247-1251 (1991); Gelderblom, et al.,
Carcinogenesis
13: 433-437 (1992)). Fusarium isolates in section Liseola produce fumonisins in culture at levels from 2 to >4000 ppm (Leslie, et al.,
Phytopathology
82: 341-345 (1992)). Isolates from maize (predominantly mating population A) are among the highest producers of fumonisin. (Leslie et al., supra). Fumonisin levels detected in field-grown maize have fluctuated widely depending on location and growing season, but both preharvest and postharvest surveys of field maize have indicated that the potential for high levels of fumonisins exists (Murphy, et al.,
J Agr Food Chem
41: 263-266 (1993)). Surveys of food and feed products have also detected fumonisin (Holcomb, et al.,
J Agr Food Chem
41: 764-767 (1993); Hopmans, et al.,
J Agr Food Chem
41: 1655-1658 (1993); Sydenham, et al.,
J Agr Food Chem
39: 2014-2018 (1991)). The etiology of Fusarium ear mold is poorly understood, although physical damage to the ear and certain environmental conditions can contribute to its occurrence (Nelson,
Mycopathologia
117: 29-36 (1992)). Fusarium can be isolated from most field grown maize, even when no visible mold is present. The relationship between seedling infection and stalk and ear diseases caused by Fusarium is not clear. Genetic resistance to visible kernel mold has been identified (Gendloff, et al.,
Phytopathology
76: 684-688 (1986); Holley, et al.,
Plant Dis
73: 578-580 (1989)), but the relationship to visible mold to fumonisin production has yet to be elucidated.
Fumonisins have been shown in in vitro mammalian cell studies to inhibit sphingolipid biosynthesis through inhibition of the enzyme sphingosine N-acetyl transferase, resulting in the accumulation of the precursor sphinganine (Norred, et al.,
Mycopathologia
117: 73-78 (1992); Wang, et al.,
Biol Chem
266: 14486 (1991); Yoo, et al.,
Toxicol Appl Pharmacol
114: 9-15 (1992); Nelson, et al.,
Annu Rev Phytpathol
31:233-252 (1993)). It is likely that inhibition of this pathway accounts for at least some of fumonisin's toxicity, and support for this comes from measures of sphinganine: sphingosine ratios in animals fed purified fumonisin (Wang, et al.,
J Nutr
122: 1706-1716 (1992)). Fumonisins also affect plant cell growth (Abbas, et al.,
Weed Technol
6: 548-552 (1992); Vanasch, et al.,
Phytopathology
82: 1330-1332 (1992); Vesonder, et al.,
Arch Environ Contam Toxicol
23: 464-467 (1992)). Kuti et al., (Abstract, Annual Meeting American Phytopathological Society, Memphis, Tenn.: APS Press 1993) reported on the ability of exogenously added fumonisins to accelerate disease development and increase sporulation of
Fusarium moniliforme
and
Fusarium oxysporum
on tomato.
Enzymes that degrade the fungal toxin fumonisin to its de-esterified form (e.g. AP1 from FB1) have been identified in U.S. Pat. No. 5,716,820, issued Feb. 10, 1998, U.S. Pat. No. 5,792,931 issued Aug. 11, 1998; and pending U.S. applications Ser. Nos. 08/888,950 and 08/888,949, both filed Jul. 7, 1997, and all hereby incorporated by reference. It is understood that AP1 as used here designates the hydrolyzed form of any fumonisin, FB1, FB2, FB3, FB4, or any other AP1-like compounds, including synthetically produced AP1 like compounds, that contain a C-2 or C-1 amine group and one or more adjacent hydroxyl groups. Plants expressing a fumonisin esterase enzyme, infected by fumonisin producing fungus, and tested for fumonisin and AP1 were found to have low levels of fumonisin but high levels of AP1. AP1 is less toxic than fumonisin to plants and probably also to animals but contamination with AP1 is still a concern (Lamprecht, et al.,
Phytopathology,
84:383-391 (1991)). The preferred result would be complete detoxification of fumonisin to a non-toxic form. Therefore enzymes capable of degrading AP1 are necessary for the further detoxification of fumonisin.
The present invention provides newly discovered polynucleotides and related polypeptides of amino polyol amine oxidase (abbreviated APAO, formerly known as AP1 catabolase, U.S. Pat. No. 5,716,820, supra, U.S. Pat. No. 5,792,931 supra; pending U.S. applications Ser. Nos. 08/888,950 and 08/888,949, supra; trAPAO is the abbreviation for a truncated, but still functional APAO), capable of oxidatively deaminating the AP1 to a compound identified as the 2-oxo derivative of AP1 or its cyclic ketal form (abbreviated as 2-OP, formerly called AP1-N1, U.S. Pat. No. 5,716,820, and U.S. Pat. No. 5,792,931 supra; pending U.S. applications Ser. Nos. 08/888,950 and 08/888,949, supra), isolated from
Exophiala spinifera
, ATCC 74269. The partially purified APAO enzyme from
Exophiala spinifera
has little or no activity on intact FB1, a form of fumonisin. However, recombinant APAO enzyme from
Exophiala spinifera
, expressed in
E. coli
, has significant but reduced activity on intact FB1 and other B-series fumonisins. APAO or trAPAO thus could potentially be used without fumonisin esterase since the amine group is the major target for detoxification. Alternatively, fumoninsin esterase and APAO (or trAPAO) can be used together for degrading toxins.
APAO is a type of flavin amine oxidase (EC 1.4.3.4, enzyme class nomeclature, see
Enzyme Nomenclature
1992, Recommendations of the Nomenclature Committee of the IUBMB on the Nomenclature and Classification of Enzymes, Academic Press, Inc. (1992)). Flavin amine oxidases are known in mammals as monoamine oxidases, where they participate in the conversion of amines involved in neuronal function. A prokaryotic flavin amine oxidase that deaminates putrescine has been described (Ishizuka et al.,
J Gen Microbiol.
139:425-432 (1993)). A single fungal gene, from Aspergillus niger has been cloned (Schilling et al.,
M

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