Ethanolaminephosphate cytidylyltransferase gene and promoter

#The present invention relates generally to the fields of genes and promoters. More particularly, the present invention relates to an isolated gene and promoter of the enzyme ethanolaminephosphate cytidylyltransferase.

#Phosphatidylethanolamine (PE) is an abundant lipid in both eukaryotic and prokaryotic cells. PE is situated primarily on the inner leaflet of the cell membrane where it interacts with inner-membrane proteins (1) or acts as a molecular chaperone and assists in proper protein folding (2). PE also plays an important role in physiological processes such as blood coagulation, platelet activation, cell signalling, membrane fusion, cell cycle progression, cell division, and apoptosis (3-9). Transfer of PE from lipoproteins to platelets induces their activation (3) and PE induces high-affinity binding sites for factor VIII and stimulates its pro-coagulant activity (4). PE is also involved in the thrombotic activity found in some cases of lupus where it further inhibits activated protein C (5). PE is a direct precursor of other lipids and provides ethanolamine moiety for anandamide (a physiological ligand for the cannabioid receptors) and glycosylphospatidylinositol (GPI) membrane anchors for a diverse group of proteins known as proteoglycans.

#Distribution of PE in membranes plays a pivotal role during cytokinesis (7). Prior to late telophase, PE becomes exposed on the cell surface at the cleavage furrow, where it regulates the movement of the actin contractile ring and plasma membrane (7). Interestingly, the surface trapping of PE causes cell arrest (10), and the appearance of PE (together with PS) on the cell surface is an early hallmark of apoptosis (8). Products of PE metabolism, fatty acids, diacylglycerols and phosphatidic acid serve a critical role as second messengers in various signalling pathways and PE is an immediate donor of phosphoethanolamine residue linking glycosylphosphatydylinositol (GPI) anchor to proteins (11-14). There exist specialized forms of PE such as plasmalogens and derivatives such as natural cannabinoid anandamide and glycosylated PE (15-17). Plasmalogens play a role in the prevention of oxidation of lipoproteins (16) and constitute a significant portion of total PE in many tissues (18,19). However, despite their relative abundance, the principal biological function of plasmalogens is not firmly established and the understanding of the regulation of their production is surprisingly limited. Their production is severely impaired in the peroxisomal disorders such as Zellweger syndrome, Refsum disease (11) and neurological disorders (21-23). Glycosylated PE is abundant in lipoproteins of diabetics and has been implicated in the promotion of atherosclerosis in those individuals (24).

#Embodiments of the present invention will now be described, by way of example only, with reference to the attached figures.

#FIG. 1 shows a schematic representation of the three known biosynthetic pathways for phosphatidylethanolamine (PE) in mammalian cells.

#FIG. 2 is an alignment of amino acid sequences of rat ET (rET), human ET (hET), and the mouse ET (mET) proteins. The alignment illustrates the ET consensus motifs MIRNG and HYGH, conserved in the entire cytidylyltransferase superfamily.

#This is the first characterization of the mouse and human CTP: phosphoethanolamine cytidylyltransferase genes. The isolated gene and promoter sequences are novel and do not match any other sequences in the mouse and human database. The mouse gene sequence is unique and distinct from the human gene sequence. The isolated gene and promoter have a number of applications within the biotechnology and pharmaceutical industries.

#The invention is based on the screening of the mouse RP23 BAC (Bacterial Artificial Chromosome) library by using the 3′-end “overgo” cDNA sequence of the mouse ET. Thirteen positive mouse ET clones were identified. The BAC clones were reamplified with different primers, and after subcloning into a PCR vector and sequencing all positive clones were identified, the mouse ET gene was reconstructed. The BAC clones encoding the full-length mouse ET gene were isolated. The murine ET gene and the 5′ flanking (regulatory, promoter) region of the human ET gene were determined and are disclosed herein. The characterization of the overall structure for the murine and human ET genes as well as the localization of cis-DNA elements for transcription factors in the 5′flanking promoter region of the human gene are disclosed.

#A gene according to the invention encoding a protein having ethanolaminephosphate cytidylyltransferase activity. The gene consists of a sequence selected from the group consisting of: (a) SEQ ID NO:1; (b) a degenerate sequence of SEQ ID NO:1 and (c) a sequence which hybridizes to the complement of SEQ ID NO:1 under stringent conditions. A portion of such a gene capable of encoding a protein having ethanolaminephosphate citidylyltransferase activity also falls within the scope of the invention. In the examples put forth herein, SEQ ID NO: 1 was isolated from mouse.