Mammalian CDP-diacylglycerol synthase

Abstract

There is disclosed cDNA sequences and polypeptides having the enzyme CDP-diacylglycerol synthase (CDS) activity. CDS is also known as CTP:phosphatidate cytidylyltransferase. There is further disclosed methods for isolation and production of polypeptides involved in phosphatidic acid metabolism and signaling in mammalian cells, in particular, the production of purified forms of CDS.

We claim:  
     
         1 . An isolated polynucleotide, comprising a polynucleotide sequence selected from the group consisting of: 
 (a) the DNA sequence of FIG. 8 and biologically active fragments thereof; and    (b) a DNA sequence which encodes the polypeptide of FIG. 8 and biologically active fragments thereof;    wherein said polynucleotide encodes a polypeptide having CDP-diacylglycerol synthase (CDS) activity.    
     
     
         2 . A method of expressing a polypeptide from the polynucleotide of  claim 1 , comprising: 
 (a) introducing into a cell the polynucleotide of  claim 1 , 
 wherein said polynucleotide is operably linked to a promoter; and  
   (b) maintaining or growing said cell under conditions that result in the expression of a polypeptide having CDS activity.    
     
     
         3 . The isolated polynucleotide of  claim 1 , wherein said polynucleotide comprises (a) the DNA sequence of FIG. 8 or (b) the DNA sequence encoding the polypeptide of FIG. 8.  
     
     
         4 . The isolated polynucleotide of  claim 3 , wherein said polynucleotide comprises the DNA sequence of FIG. 8.  
     
     
         5 . The isolated polynucleotide of  claim 3 , wherein said polynucleotide comprises the DNA sequence encoding the polypeptide of FIG. 8.  
     
     
         6 . An isolated polynucleotide, comprising a polynucleotide sequence that hybridizes under high stringency conditions to the DNA sequence selected from the group consisting of: 
 (i) the complement of the coding region of the DNA sequence of (a) of claim  3 ; and    (ii) the complement of the DNA sequence of claim (b) of claim  3 ;    wherein said polynucleotide encodes a protein having CDS activity.    
     
     
         7 . An isolated polynucleotide, comprising a polynucleotide sequence having at least 85 percent identity to the DNA sequence selected from the group consisting of: 
 (i) the coding region of the DNA sequence of (a) of claim  3 ; and    (ii) the DNA sequence of (b) of claim  3 ;    wherein said polynucleotide encodes a protein having CDS activity.    
     
     
         8 . The isolated polynucleotide of  claim 1 , wherein said polynucleotide encodes a polypeptide having an intact CDS N-terminal region.  
     
     
         9 . An isolated polypeptide, comprising the amino acid sequence of FIG. 8 and biologically active fragments thereof, wherein said polypeptide has CDS activity.  
     
     
         10 . The isolated polypeptide of  claim 9 , wherein said polypeptide comprises the amino acid sequence of FIG. 8.  
     
     
         11 . An isolated polypeptide, comprising an amino acid sequence having 85 percent sequence identity to the amino acid sequence of FIG. 8, wherein said polypeptide has CDS activity.  
     
     
         12 . An isolated polypeptide, comprising the polypeptide encoded by the polynucleotide of  claim 6 .  
     
     
         13 . An isolated polypeptide, comprising the polypeptide encoded by the polynucleotide of  claim 7 .  
     
     
         14 . A method for screening one or more compounds to determine whether said one or more compounds increases or decreases CDS activity, comprising: 
 (a) contacting the polypeptide of  claim 9  with one or more substrates for said polypeptide and with said one or more compounds; and    (b) measuring whether the CDS activity of said polypeptide is increased or decreased by said one or more compounds.    
     
     
         15 . The method of  claim 14 , wherein said one or more compounds is selected from a combinatorial chemical library.  
     
     
         16 . The method of  claim 9 , wherein said polypeptide contains an intact CDS N-terminal region.