Title of Invention


Abstract The present invention relates to the isolation of a nucleic acid sequence, the product of which, confers stress tolerance against salinity, drought, high and low temperatures and is also a useful molecule for drugs on account of its antioxidant properties, which are of immense benefit in the detoxification of alkyl hydroperoxide.
Full Text

Peroxiredoxins, which have been identified in all groups of organisms, constitute a phylogenetically old group of enzymes with catalytic function in the detoxification of cell-toxic peroxides. Within the peroxiredoxins, four clusters of related proteins are distinguished: 1-Cys peroxiredoxins (1-CP), 2-Cys peroxiredoxins (2-CP), YLR109-related peroxiredoxins (type II-Prx), and bacteriferritin-associated proteins (Prx Q). All Peroxiredoxins are characterized by one or two cysteine residue(s), located in a conserved structural environment of the protein and are central for the catalytic reaction.
The functional 2-Cysteine peroxiredoxin is a homodimer with two reduced cysteine residues per subunit. During the catalytic cycle, the peroxide substrate is reduced to the corresponding alcohol and the cysteine residues oxidized to a disulphide bridge (Fig. 1). The regeneration of catalytically active 2-Cysteine peroxiredoxin depends on reduction of the disulphide bridge. In most eukaryotic organisms, thioredoxin or thioredoxin-related proteins such as trypathionin act as electron donors. (Journal of Experimental Botany, Vol. 53, No. 372, pp. 1321-1329, May 15,2002)
Fig. 1. Simplified scheme of peroxide reduction by Prx and regeneration of oxidized Prx through an appropriate electron donor like Trx or GSH.

Peroxiredoxins reduce peroxide substrates to the corresponding alcohol. In the 1-CP, 2-CP, type II-prx, and prx Q, a Cys-residue in conserved structural environment constitutes the amino acid residue, essential for the catalytic activity with the peroxide substrates. During the catalytic cycle with two peroxides two disulphide bonds are successively formed with intermediate modification of one cysteine residue to cysteine-sulphenic acid (Cys-OH) and subsequent release of H2O. Thus, three different conformational states of the 2-Cysteine peroxiredoxin exist and can be detected in non-reducing denaturing gels as distinct bands

in electrophoretic mobility under non-reducing conditions represents a convenient assay for reactivity with peroxide substrates (Fig. 2*). Oxidation of the 2-Cysteine peroxiredoxin also occurs during exposure to dissolved oxygen. The fully oxidized 2-CP loses its enzymatic activity and must be regenerated. For many peroxiredoxins it has been established that thioredoxins serve as the electron donor for the regeneration of the active form. In chloroplasts, thioredoxin f and m function in the regulation of redox-sensitive target proteins and could be involved in the regeneration of oxidized 2-Cysteine peroxiredoxin.

The homodimer can successively reduce two peroxides substrates, concomitantly two disulphide bridges are formed. Regeneration requires reduction of the disulphides. Electron donor may be thioredoxin. The three distinct redox states of the 2-CP can be visualized in non-reducing denaturing polyacrylamide gels. The fully reduced 2-CP disintegrates into monomers of about 25 kDa under denaturing conditions, the dimers linked by one or two disulphide bridge(s) show a distinct electrophoretic mobility.
Peroxiredoxins reduce a broad range of alkyl hydroperoxide substrates including short- and long-chain alkyl hydroperoxide, phospholipid peroxides and cholesterol peroxide (Nogoceke et ah, 1997+; Hillas et al, 2000*). For the time being, it can be assumed that plant Prx also shows little discrimination between various alkyl hydroperoxide substrates and reduce diverse peroxides. Alkyl hydroperoxides are produced by spontaneous chemical reactions of unsaturated organic substrates with reactive radicals such as OH ,or by enzymatic reaction of polyunsaturated lipids with lipoxygenases. Subsequently, alkyl hydroperoxides may initiate radical chain reactions and cause membrane destruction. Peroxides need to be eliminated to avoid severe damage of the cells. In addition to detoxification by non-enzymatic antioxidants, two types of enzymes exist in the chloroplasts which are suggested to catalyse the reduction of alkyl hydroperoxides (Baier and Dietz, 1999*a, b+): (i) The phospholipid hydroperoxide glutathione peroxidase (PHGPx) (Mullineaux et al, 1998*) which reacts with peroxide substrates by concomitant oxidation of a cysteine residue. PHGPx is reactivated with reduced glutathione (Eshdat et a/., 1997+). (ii) The 2-CPs which have a similar substrate spectrum as PHGPxs and are regenerated by thioredoxin. Analysis of transgenic Arabidopsis with reduced 2-CP contents suggest that PHGPx cannot substitute for decreased 2-CP activity (Baier and Dietz, 1999+fc; Baier et ah, 2000*). (i) Antisense suppression of 2-CP caused increased damage of chloroplast proteins and impaired photosynthesis, (ii) Increased oxidation of the

ascorbate pool and up-regulation of Apx and MDAR transcript level and activity indicated a major disturbance of antioxidant metabolism in the mutant plants, despite the fact that (iii) the PHGPx-transcript level, which was at least 100-fold lower than the 2-CP transcript level, was unchanged in the antisense mutants. A distinct sub-organellar compartmentalization may provide the explanation for the lack of interchangeability of PHGPx and 2-CP in the chloroplast. The PHGPx has been reported to be a soluble enzyme located in the stroma (Mullineaux et a/., 1998*) whereas the 2-CP is preferentially attached to the thylakoid membrane.

So far, plant 2-cysteine peroxiredoxins have been cloned from spinach (EMBL Ace. No. X94219), barley (EMBL Ace. No. Z34917), Arabidopsis thaliana(EMBL Ace. No. Y10478), wheat (EMBL Ace. No. Ab000405), rice (EMBL Ace. No. D48223), rye (EMBL Ace. No. Af076920) and from the liverwort Riccia fluitans (EMBL Ace. No. AJ005006). Work with transgenic organisms has proven the antioxidant function of plant 2-cysteine peroxiredoxins in vivo:
• Deletion of the endogenous alkyl hydroperoxide reductase AhpC increases the sensitivity of E. coli to cumene hydroperoxide. When the deletion mutant is complemented with the plant 2-CP gene, E. coli is less susceptible to alkyl hydroperoxide than the wild type.
• Arabidopsis plants expressing the 2-cysteine peroxiredoxins gene in antisense suffer from increased photoinhibition at elevated light intensities and contain lower amounts of chloroplast proteins during early phases of development. Photosynthetic activity is impaired during the young stages of rosette development. The photo-oxidative stress of the transgenic plants is compensated partly by increased superoxide dismutase and guajacol peroxidase activities. Later, during development, the 2-cysteine peroxiredoxins leaf content approaches that of wild-type plants and the obvious antisense phenotype disappears, indicating a high stability and low turnover of 2-cysteine peroxiredoxins protein.
Prior Art - Expressing the 2-Cysteine peroxiredoxin gene in Arabidopsis thaliana plants in antisense orientation has revealed a decreased growth rate and impaired photosynthesis, and, has thereby proved the role of the gene in antioxidative defence. Reports on this molecule suggest that the human malarial parasite, Plasmodium falciparum can act as a terminal peroxidase of the parasite, Thioredoxin reductase system.
Literature has revealed that 2-Cysteine peroxiredoxin constitutes a ubiquitous group of peroxidases that reduce the toxicity in cells. The photosynthetic machinery during leaf development requires a high level of 2-Cysteine peroxiredoxin to protect it from oxidative damage.
US patent no. 6002068 describes a method for conferring insect resistance to a monocot using a perioxidase coding sequence.
US patent no. 6278041 describes an anionic peroxidase gene sequence isolated from Nicotiana tomentisiformis. In addition, synthetic Nicotiana tomentisiformis and Nicotiana sylvestris peroxidase gene sequences were optimised for expression in plants. The peroxidase gene sequences may be expressed in transgenic plants to control insects.
US patent application no. 20020083492 relates to a plant, into which DNA encoding a basidiomycete-derived peroxidase is transferred, said DNA being expressed therein, and to a method for decomposing and removing hazardous chemicals in an environment effectively by using the plant.

US patent application no. 20020100081 This invention provides a novel method to render thermotolerance to a plant. In the concrete, this method provides peroxisomal ascorbate peroxidase gene of barely, a novel gene induced by high temperature stress. Moreover, this invention provides a transgenic plant exhibiting resistance to high-temperature stress, produced by incorporating said gene.

Gloriosa superba belongs to the family Liliaceae (sub-family-Wurmbaeoideae). It yields different kinds of tropolone alkaloids, which are of medicinal importance, the significant ones being colchicine, 3-democolchicine and colchicoside.
Gloriosa superba is a valuable tropical medicinal plant and almost the whole of it is used in the indigenous system of medicine. Due to poor seed germination, the plant is propagated vegetatively through corms. Indiscriminate overexploitation of Gloriosa for diverse medicinal usage has endangered its very survival.
The underground root stocks have been utilized in the Indian system of medicine viz. ayurveda, since ancient times as an antihelminthic, anti inflammatory and antileprotic. It is useful in treating dermatosis, piles, chronic ulcers, colic pain and as a cataplasm in neuralgic pain. The white starchy powder that emanates from the tuber is of great use in treating gonorrhoea and the juice obtained from the leaves is used to kill lice in the hair and scalp. The underground stocks have been mainly used as an abortifacient.
Due to poor seed germination, the plant is propagated vegetatively through corms. Indiscriminate overexploitation of Gloriosa for diverse medicinal usage has endangered its very survival.
Colchicine an important alkaloid, is often used to treat gout and acute gouty arthritis and is known to relieve pain effectively. The mode of action of colchicine in gout is unknown, but it is believed to decrease lactic acid production by leukocytes thereby decreasing urate crystal deposition and subsequently reducting in phagocytosis, with a decrease in the inflammatory response. It is also known to alter the neuromuscular functions, intensify gastrointestinal activity by neurogenic stimulation, increase sensitivity to central depressants, heighten response to symathomimetic compounds and depress the respiratory center.
It is known to play an important role in breeding by inducing polyploidy. Colchicine is also known to inhibit mitosis, interfere with the orientation of fibrils and has been used in the treatment of cancer. Colchicine itself is too toxic for human use as an antitumor drug and the use of its derivatives, which are less toxic viz, democolcicine, trimethylcolchicine acid, methylester, 2-demethyl and 3-demethyl thiocolchicine, have, been evaluated as anti leukemic agents.
Colchicine and colchicoside, which were earlier, isolated from Colchicum autumnale L, but, with the discovery of colchicine in Gloriosa superba, the latter has become a potential commercial source for the pharmaceutical industry and also on account of their presence in appreciable quantities in seeds and other parts of the plant. Bellet and Gaignault (1985) compared the relative colchicine content of the genera Colchicum (the traditional source of Colchicine) and Gloriosa on the dry mass basis and they yielded 0.9% and 0.82% respectively.
Colchicine, a structurally more intriguing alkaloid, is the active principle of several species. The structure of colchicine has been a long-standing problem with chemists, and, from labeling studies, it has been proved that the metabolic paths of phenylalanine and tyrosine follow separate lines in plants. The two amino acids give, a phenylethylisoquinoline derivative which undergoes a directed p,p-phenol coupling to produce the homomorphinone

skeleton. The phenylalanine-cinnamic pathway has been shown to be involved in ring A and the carbon atoms C-5, C-6 and C-7 (Herbert and Knagg,1986). The aromatic ring and the benzyl carbon atom of tyrosine is involved in the genesis of troplone ring.
The phenylethylisoquinoline alkaloids isolated from the six genera of Liliaceae characteristically contain the C6-C3-N-C2-C6 unit. Their biosynthesis follows aclose parallel with the corresponding alkaloids of the benzoisoquinoline group. The C6-C2 fragment originates from tyrosine via dopa and dopamine and the C6-C3 fragment originates from phenylalanine, which, conventionally is transformed into 4-hydroxydihydrocinnamaldehyde. The homoapoporhine kreysigine is formed by the condensation of dopamine with a methoxylated dihydrocinnamaldehyde to autumnaline, which subsequently, undergoes phenol oxidation and further, methylation, solvolysis and by a rearrangement of these, colchicine is produced.
Cysteine 2-peroxiredoxin could be a potential source for drug discovery programmes since it possesses antioxidant properties. This molecule can also be implicated in the detoxification of alkyl hydroperoxides.
Oxidative stress, which is induced by a variety of physiological and pathological conditions, produces reactive oxygen species that damage DNA and most other biological macromolecules. To prevent such cellular damage, living organisms have evolved a defence system involving several antioxidant enzymes.
We have been able to generate a cDNA clone from a pool of mRNA, isolated from a 13 day old plantelet of Gloriosa superba, the function of which has been found to be associated with antioxidative detoxification.
1. Total RNA was extracted from the 13 day old actively growing apical region of the Gloriosa superba plant following the Trizol protocol (Life Technologies).
2. Subsequently, the mRNA was isolated from the total RNA following Oligotex mRNA Batch Protocol (Genetix)
3. A cDNA library was constructed by making use of the GIBCOBRL Superscript Plasmid System with the Gateway Technology for the cDNA Synthesis and Cloning Kit.
4. The cDNA clones obtained were screened and selected for sequencing and subjected to a data base search in order to ascertain their identity.
5. Functional details of the clones were collected and one cDNA clone was designated as cDGsCo04Hl 1, which was predicted to be a homologue of a 2-Cys Peroxiredoxin.

We claim
1. A method of enhancing the capabihty of a host plant to detoxify reactive oxygen
species by expressing 2-Cysteine peroxiredoxin nucleotide sequence and generating a
whole generation of transgenic plants that confer environmental stress tolerance,
disease resistance and possess anti oxidant properties thereby imparting immense
value as a novel therapeutic molecule.
2. A claim as in claim 1, wherein the transgenic plants could apply to all varieties of
3. A claim as in claim 1, wherein the transgenic plants confer increased tolerance to
environmental stress conditions such as drought, salinity, ultra violet radiation, heat
and cold.
4. A claim as in claim 3, wherein the markers used in the technology will play a
significant role in seed purity testing for disease tolerance.
5. A claim as in claim 1, whereby 2-Cysteine peroxiredoxin will play a useful role as a
novel therapeutic molecule.
6. A claim as in claims 1 and 5, wherein the 2-Cysteine on account of its antioxidant
properties, will immensely benefit the detoxification of alky 1 hydroperoxide.





737-che-2003-form 1.pdf

Patent Number 241450
Indian Patent Application Number 737/CHE/2003
PG Journal Number 28/2010
Publication Date 09-Jul-2010
Grant Date 05-Jul-2010
Date of Filing 16-Sep-2003
# Inventor's Name Inventor's Address
PCT International Classification Number G06F 17/00
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA