|Title of Invention||
PROCESS OF ISOLATING NUCLEIC ACID SEQUENCE DIACYLGLYCEROL KINASE FROM TERMINALIA ARJUNA & ITS USES THEREOF
|Abstract||The present invention relates to the isolation of a nucleic acid sequence, the product of which, confers intercellular messengers in signal transduction pathway. The invention also relates to the generation of a cDNA clone from the pool of mRNA, isolated from the mature leaves of Terminalia arjuna, the function of which, confers signal transduction molecule and thus, acts as a messenger.|
and other cells. Like interior walls of houses, biological membranes are structurally organized barriers that compartmentalize and organize the cell's intracellular components.
Biological membranes, however, are not impervious walls. Instead, they are highly selective permeable barriers, which regulate the quality and quantity of molecules, which are allowed to pass through the membrane. The cell membrane, for example, tightly regulates the amount of water, ions and sugar, which can pass into the cell.
One class of lipids, which is abundant in all biological membranes, is phosphoglycerides. Phosphoglycerides are comprised of a glycerol (a three-carbon alcohol) backbone, two fatty acid chains (long hydrocarbon molecules), and a phosphate. The simplest phosphoglyceride that can be formed is phosphatidic acid. Phosphatidic acid has two fatty acid chains esterified to the hydroxyl groups at the C-1 and C-2 positions of glycerol, respectively. The C-3 hydroxyl group of glycerol is esterified to phosphoric acid. While phosphatidic acid is not a major component of biological membranes, it is a key intermediate in the formation of structurally related phosphoglycerides such as phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol, which has a sugar moiety attached to the phosphate.
Second, fatty acid-containing lipids are an energy source for cells and organisms. Fatty acids in a series of biological reactions are oxidized by certain cells to yield large amounts of energy necessary to carry out essential biological functions. Fatty acids used for fuel are stored as triacylglycerols (which are sometimes referred to as "triglycerides"), or neutral fats. Like phosphatidic acids, triglycerides have a glycerol backbone. Rather than having two fatty acid and a phosphate group, however, triglycerides contain three fatty acid chains.
Triglycerides are an efficient way to store large quantities of energy, and thus are the major energy reservoir in humans and other mammals. The complete oxidation of a typical fatty acid yields approximately 9 kcal/g, as compared to about 4 kcal/g for proteins and carbohydrates. Moreover, unlike carbohydrate energy stores, triglycerides are anhydrous (i.e., do not contain water). Consequently, a gram of triglycerides contains more than six times the energy of one gram of carbohydrate. Taken together, triglycerides account for about 80% of all the energy of an average individual.
Finally, lipids participate in cell-cell communication, differentiation and proliferation. Normal development and function in living organisms requires interactions between cells and the molecules in the surrounding environment. One way cells communicate is via molecules, called transmembrane proteins, that span the cell's biological membrane. When the portion of the transmembrane protein which is outside of the cell encounters specific molecules in the surrounding environment, it undergoes conformational changes that trigger a biological cascade inside the cell.
The binding or interaction of a molecule in the environment with a transmembrane protein frequently activates a membrane-bound enzyme called phospholipase C. The activation of phospholipase C is at the center of many major biological events. For example, the activation of phospholipase C is correlated with cell proliferation. Vasopressin, prostaglandin F2, and bombesin, which stimulate cell proliferation,
stimulate phospholipase C. In addition, phospholipase C plays a role in activation of T lymphocytes of the immune system and fertilization of eggs.
Phospholipase C exerts its biological effects by catalyzing a reaction, which cleaves the sugar moiety of the cell membrane lipid phosphatidylinositol 4,5 bisphosphate. The reaction releases diacylglycerol (DAG) and inositol triphosphate. Diacylglycerol and inositol triphosphate, referred to as second messengers, in turn, activate other molecules within the cell. Diacylglycerol, for example, activates an enzyme called protein kinase C (PKC), which is central to numerous biological processes, including the regulation of cell growth and differentiation.
DAG is at the heart of lipid-mediated biological events. See U.S. Patent application Ser. No. 08/841,483 filed Apr. 22, 1997. Diacylglycerol is a precursor to phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol, which are indispensable components of biological membranes. In addition, diacylglycerol is a precursor to triglyceride biosynthesis and, therefore, is central to energy stores of organisms. Finally, diacylglycerol is a second messenger which binds and activates protein kinase C, leading to numerous biological events.
The proper regulation of diacylglycerol in cells, therefore, is critical for proper biological function. Abnormally high or low levels of diacylglycerol would be predicted to alter the lipid biosynthesis and the activity of enzymes that depend on diacylglycerol, like PKC.
Diacylglycerol kinase (DGK), which catalyzes phosphorylation of DAG to phosphatidic acid (PA), is thought to be a key enzymes in the regulation of DAG levels and, as a result, to be responsible for attenuating the activation of PKC. For example, a constitutively elevated level of DAG (leading to activated PKC) is common in transformed cells, and experimental overexpression of DGK.alpha. decreased the elevated DAG level in ras-transformed fibroblasts. T. Fu et al. (1992), FEBS Lett.307:301-304. This type of experiment suggests that conversion of DAG to PA suppresses a mitogenic signal, but this conclusion is complicated by the fact that PA itself may be mitogenic. W. Moolenaar et al. (1986), Nature 323:171-173. PA has been implicated in the regulation of DNA synthesis, in the induction of c-myc, c-fos, and platelet-derived growth factor; in cAMP formation; and in modulating the activity of n-chimaerin and NFl. Thus, since both DAG and PA can act as second messengers, their interconversion is likely to be tightly regulated.
This cDNA clone is of potential value in plant biotechnology programmes by aiding in manipulating the signal transduction pathway. The gene could be a potential source in aiding drug discovery programmes.
1. Total RNA was extracted from mature leaves of Terminalia arjuna plant.
2. Subsequently, mRNA was isoated from the total RNA.
3. A cDNA library was constructed with the aid of the GIBCOBRL Superscript Plasmid System with the Gateway Technology for the cDNA Synthesis and Cloning kit,
4. The clones were screened and selected for sequencing and subjected to a data-base search to ascertain their identity.
5. Functional details of the clones were collected and one cDNA clone designated as cDTaML01C07, was predicted to be a homologue of Diacylglycerol kinase.
1. The nucleic acid sequence and/or a fragment of the same thereof encoding a
diacylglycerol kinase enzyme isolated from the mature leaves of Terminalia
arjuna, the function of which, confers signal transduction molecule and thus, acts
as a messenger.
2. The method of transforming plants with diacylglycerol kinase nucleic acid
sequence, the product of which, confers intercellular messengers in signal
3- A claim as in claim 1, wherein the transgenic plants could apply to all varieties of plants.
4. A construct including a nucleic acid or nucleic acid fragment according to
5. A vector including a nucleic acid or nucleic acid fragment according to claim
6. A vector according to claim 5 including a promoter, with the said promoter,
nucleic acid or nucleic acid fragment being operatively linked.
7. The diacylglycerol kinase gene isolated from the mature leaves of Terminalia
arjuna possessing potential value in plant biotechnology programmes by
aiding in manipulating the signal transduction pathway.
8. The diacylglycerol kinase gene isolated from the mature leaves of Terminalia
arjuna possessing a potential source in aiding drug discovery programmes.
|Indian Patent Application Number||774/CHE/2003|
|PG Journal Number||30/2010|
|Date of Filing||24-Sep-2003|
|Name of Patentee||Avestha Gengraine Technologies Pvt Ltd|
|Applicant Address||'DISCOVERER' 9TH FLOOR, UNIT 3, INTERNATIONAL TECH PARK WHITEFIELD ROAD BANGALORE 560 066|
|PCT International Classification Number||C12N15/00|
|PCT International Application Number||N/A|
|PCT International Filing date|