Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or...
Reexamination Certificate
2000-07-12
2004-01-13
Bui, Phuong T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
C435S006120, C435S069100, C435S183000, C435S410000, C435S419000, C435S252300, C435S320100, C530S350000, C530S370000, C536S023200, C536S023600, C536S024100, C800S295000
Reexamination Certificate
active
06677502
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 metabolism proteins in plants and seeds.
BACKGROUND OF THE INVENTION
Gibberellic acid (GA) is an important regulator (phytohormone) of plant development. Gibberellic acid has been shown to stimulate elongation in the internodes of stems and to play roles in flower and fruit development. Identification and characterization of genes involved in GA biosynthesis will permit genetic engineering methods aimed at modulating levels of GA in plants which will in turn allow for better control of plant stature, fertility and plant development in general (see World Patent Publication No. WO 95/35383).
Gibberellic acid is synthesized from isoprenoid geranylgeranyl diphosphate (GGDP), beginning with the conversion of GGDP to copalyl diphosphate (CDP). Copalyl diphosphate is then converted to GA
12
-aldehyde which in turn can be converted to a number of different gibberellins required for normal plant development. For example GA-20 oxidase catalizes the conversion of GA12 to GA9. A key enzyme in the synthesis of gibberellin is dioxygenase which appears to play a role in the conversion of GA
12
-aldehyde to GA
9
and GA
25
. Because GA
12
-aldehyde dioxygenase appears to catalyze key steps in the synthesis of GA it is a target enzymes that may be manipulated to control GA levels. Ent-Kaurene synthase A (KSA) catalyzes the conversion of GGDP to CDP, which is subsequently converted to ent-kaurene by ent-kaurene synthase B (KSB) (Yamaguchi et al. (1996)
Plant J.
10(2):203-213). Gibberillin 3-beta-hydroxylase catalizes the conversion of GA20 to GA1 a major gibberellin that is involved in controlling stem elongation. These enzymes catalyze key steps in the synthesis of GA and thus provide target enzymes that may be manipulated to control GA levels.
Thus there is a great deal of interest in identifying genes that encode proteins that may be used to control plant developmental. Accordingly, the availability of nucleic acid sequences encoding all or a substantial portion of a GA dioxygenase would facilitate studies to better understand plant development and provide tools to genetically engineer improved developmental properties in plants.
Riboflavin is the precursor to essential electron transport chain components and redox coenzymes such as flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Humans, unlike plants and bacteria, are incapable of synthesizing riboflavin (vitamin B
2
) from GTP, and must obtain this compound through their diet. Thousands of tons of riboflavin are produced each year as additives for food and animal feed (Bacher et al. (1997)
Methods Enzymol
280:382-389). Historically, riboflavin has been made via chemical synthesis, however recent advances in biotechnology have enabled industrial production using yeast and bacteria (Humbelin et al. (1999)
J Indust Micro
&
Biotech
22:1-7). The biologically synthesized riboflavin is cheaper to produce, and the process is better for the environment.
Several enzymatic steps are required to take GTP to riboflavin. The first step in bacteria is catalyzed by a GTP cyclohydrolase II activity which takes GTP to 2,5-iamino-6-ribosylamino-4(3H)-pyrimidinone 5′-phosphate. The enzyme performing this step is encoded by the ribA gene. RibA has two enzymatic activities, the above mentioned cyclohydrolase and a 3,4-dihydroxy-2-butanone 4-phosphate synthase activity that takes ribulose 5-phosphate to L-3,4-dihydroxy-2-butanone-4-phosphate, which is combined to a pathway intermediate, to form 6,7-dimethyl-8-ribityllumazine, the penultimate intermediate to riboflavin. The second step in the pathway is encoded by ribG a riboflavin-specific deaminase.
Studies using riboflavin over-producing
Bacillus subtilis
strains, have led to the conclusion that the GTP cyclohydrolase II/3,4-dihydroxy-2-butanone 4-phosphate synthase enzyme is rate-limiting for high-level riboflavin accumulation (Humbelin et al. (1999)
J Indust Micro
&
Biotech
22:1-7). Increasing the copy number of the ribA gene in these strains results in improved riboflavin productivity. The pathways leading to riboflavin biosynthesis are largely conserved between plants and bacteria. Therefore, the potential exists for improving the riboflavin content in crop plants, thus reducing the need for vitamin supplementation in food.
The present invention describes the identification and utility of GTP cyclohydrolase II/3,4-dihydroxy-2-butanone 4-phosphate synthase sequences from corn, rice, soybean, and wheat. Also disclosed are sequences from corn, rice, and wheat that encode riboflavin-specific deaminases. It is believed that modulation of these activities through over-expression, under-expression, or mutation will lead to altered levels of riboflavin in plants.
Hormones in animal systems and phytohormones in plants control many metabolic processes. Phytohormones differ in their structure and specific actions compared to animal hormones, though the signal transduction mechanisms involved may be similar in plants and animals. Phytohormones affect shoot elongation, stem elongation, root growth, seed dormancy, fruit ripening, leaf senescence and morphogenesis, disease resistance (Hoffman et al. (1999)
Plant Physiol
119:935-949), to name a few.
Among the phytohormones that have been studied so far, ethylene is the simplest in terms of chemical structure. Its effects, however, are far-ranging, affecting seed dormancy, fruit ripening and abscission, flower development, leaf senescence, adventitious root formation, and shoot and root growth and differentiation. Ethylene is synthesized from methionine via three enzymatic steps. Methionine is converted to S-adenosyl-methionine (SAM) by SAM synthetase, which is then converted to 1-aminocyclopropane-1-carboxylic acid (ACC) by ACC synthase. Finally, ACC oxidase acts on ACC to yield ethylene. The genes encoding these enzymes have been cloned, and transgenic approaches based on these genes have been attempted to control ethylene levels, and consequently fruit ripening. Using antisense technology, ACC synthase or ACC oxidase activities have been reduced in transgenic plants leading to inhibition of fruit ripening (Hamilton et al. (1990)
Nature
346:284-287; Oeller et al. (1991)
Science
254:437-439).
The mechanisms by which ethylene regulates plant development however are yet to be clearly defined. Ethylene-insensitive mutants and constitutive ethylene response mutants, principally in Arabidopsis, have been valuable in outlining the ethylene response pathway (Kieber (1997)
Annu. Rev. Plant Physiol. Plant Mol. Biol.
48:277-296). The isolation of the genes affected in these mutants indicates the involvement of a protein kinase cascade in ethylene signaling. CTR1, a negative regulator of the ethylene response encodes a serine/threonine kinase that is most similar to the Raf family of protein kinases (Kieber et al. (1993)
Cell
72:427-441). ETR1 in which dominant mutations lead to defective ethylene responses encodes a protein that is similar to bacterial two-component histidine kinases (Chang et al. (1993)
Science
262:539-544). It most probably serves as an ethylene receptor/ethylene response factor since etr1 mutant seedlings bind ethylene at reduced levels compared to wild-type (Bleecker et al. (1988)
Science
241:1086-1089), the ETR1 protein has been shown to bind ethylene (Schaller and Bleeker (1995)
Science
270:1809-1811), and genetic epistasis analysis puts ETR1 the start of the ethylene response pathway (Kieber et al. (1993)
Cell
72:427-441). U.S. Pat. Nos. 5,689,055 and 5,824,868 describe the Arabidopsis ETR1 gene, its tomato homologs, and their use in generating transgenic plants with modified response to ethylene.
ETR1 belongs to a small gene family in Arabidopsis, and at least one ETR1 homolog in Arabidopsis, ERS, has already been cloned (Hua et al. (1995)
Science
269:1712-1714). Homologs in other species like rice and tomato have been isolated as well (Wilki
Allen Stephen M.
Anderson Shawn L.
Caimi Perry G.
Famodu Omolayo O.
Fang Yiwen
Bui Phuong T.
E.I. du Pont de Nemours and Company
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