Method for identifying modulators gene expression

Abstract

A method of cloning mammalian genes encoding proteins which can function in microorganisms, particularly yeast, and can modify, complement, or suppress a genetic defect associated with an identifiable phenotypic alteration or characteristic in the micro-organism. It further relates to mammalian genes cloned by the present method, as well as to products encoded by such genes and antibodies which can bind the encoded proteins. More specifically, the present invention relates to a method of cloning mammalian genes which encode products which modify, complement or suppress a genetic defect in a biochemical pathway in which cAMP participates or in a biochemical pathway which is controlled, directly or indirectly, by a RAS protein, to products (RNA, proteins) enocded by the mammalian genes cloned in this manner and to antibodies which can bind the encoded proteins.

1 . A method of cloning, in a genetically altered microorganism, a mammalian gene which is capable of modifying a phenotypic alteration associated with a genetic alteration in the microorganism, comprising the steps of: 
 a) providing mammalian cDNA in an expression vector capable of expressing the mammalian cDNA in the genetically altered microorganism;    b) introducing the expression vector into the genetically altered microorganism, thereby producing genetically altered microorganisms containing the expression vector;    c) maintaining genetically altered microorganisms containing the expression vector under conditions appropriate for growth of genetically altered microorganisms; and    d) identifying genetically altered microorganisms in which the phenotypic alteration associated with the genetic alteration in the microorganism is modified.    
     
     
         2 . The method of  claim 1  further comprising transforming  E. coli  with cDNA present in genetically altered microorganisms of step (d).  
     
     
         3 . A method of cloning a mammalian gene which, when expressed in genetically altered yeast cells, modifies a phenotypic alteration associated with a genetic alteration in the yeast cells, comprising the steps of: 
 a) providing mammalian cDNA in an expression vector capable of expressing the mammalian gene in yeast;    b) introducing the expression vector of step (a) into genetically altered yeast cells, thereby producing genetically altered yeast cells containing the expression vector;    c) maintaining genetically altered yeast cells containing the expression vector under conditions appropriate for growth of genetically altered yeast cells; and    d) identifying genetically altered yeast cells in which the phenotypic alteration associated with the genetic alteration in the yeast cell is modified.    
     
     
         4 . The method of  claim 3  further comprising transforming  E. coli  with cDNA present in genetically altered yeast cells of step (d).  
     
     
         5 . The method of  claim 3  wherein the genetic alteration is associated with activation or attenuation of a biochemical pathway in which cAMP participates.  
     
     
         6 . The method of  claim 3  wherein the genetic alteration is a mutation in a gene encoding a RAS protein.  
     
     
         7 . The method-of  claim 6  wherein the genetic alteration is an activated RAS2 va119  gene of  S. cerevisiae.    
     
     
         8 . The method of  claim 7  wherein the phenotypic alteration associated with a genetic alteration is selected from the group consisting of: heat shock sensitivity, nitrogen starvation, failure to synthesize normal amounts of glycogen, failure to grow on acetate and failure to sporulate.  
     
     
         9 . The method of  claim 3  wherein the genetically altered yeast cells are genetically altered  S. cerevisiae  or genetically altered  S. pombe.    
     
     
         10 . The method of  claim 5  wherein the genetic alteration is a disruption of the PDE1 and of the PDE2 genes of  S. cerevisiae.    
     
     
         11 . The method of  claim 6  wherein the genetic alteration is a disruption of the ras1 gene of  S. pombe  or an activated allele of the ras1 gene of  S. pombe.    
     
     
         12 . Isolated DNA encoding a cAMP phosphodiesterase obtained by the method of  claim 1 .  
     
     
         13 . Isolated DNA having the nucleotide sequence of  FIG. 4 .  
     
     
         14 . Isolated protein encoded by the DNA of  claim 13 .  
     
     
         15 . Isolated DNA having the nucleotide sequence of  FIG. 5 .  
     
     
         16 . Isolated protein encoded by the DNA of  claim 15 .  
     
     
         17 . Isolated DNA having the nucleotide sequence of  FIG. 6 .  
     
     
         18 . Isolated protein encoded by the DNA of  claim 17 .  
     
     
         19 . Isolated DNA having the nucleotide sequence of  FIG. 7 .  
     
     
         20 . Isolated protein encoded by the DNA of  claim 19 .  
     
     
         21 . Isolated protein encoded by a nucleotide sequence which hybridizes to DNA having a nucleotide sequence selected from the group consisting of: 
 a) the nucleotide sequence of  FIG. 4 ;    b) the nucleotide sequence of  FIG. 5 ;    c) the nucleotide sequence of  FIG. 6 ; and    d) the nucleotide sequence of  FIG. 7 .    
     
     
         22 . A method of identifying a chemical agent which inhibits a mammalian gene which, when expressed in a genetically altered microorganism, modifies a phenotypic alteration associated with a genetic alteration in the microorganism, comprising the steps of: 
 a) expressing the mammalian gene in a genetically altered microorganism, thereby modifying the phenotypic alteration associated with the genetic alteration;    b) contacting the genetically altered microorganism of step (a) with a chemical agent to be assayed, under conditions appropriate for phenotypic assay; and    c) determining whether the phenotypic alteration associated with the genetic alteration modified in step (a) is reversed, wherein reversal of the phenotypic alteration is indicative of a chemical agent which inhibits the mammalian gene.    
     
     
         23 . A method of identifying a chemical agent which inhibits a mammalian gene which, when expressed in genetically altered yeast cells, modifies a phenotypic alteration associated with a genetic alteration in the yeast cells, comprising the steps of: 
 a) expressing the mammalian gene in genetically altered yeast cells in which the product encoded by the mammalian gene is not expressed, thereby modifying the phenotypic alteration associated with the genetic alteration;    b) contacting genetically altered yeast cells of step (a) with a chemical agent to be assayed, under conditions appropriate for phenotypic assay; and    c) determining whether the phenotypic alteration associated with the genetic alteration modified in step (a) is reversed, wherein reversal of the phenotypic alteration is indicative of a chemical agent which inhibits the mammalian gene.    
     
     
         24 . The method of  claim 23 , wherein the genetic alteration is associated with activation or attenuation of a biochemical pathway in which cAMP participates.  
     
     
         25 . The method of  claim 23 , wherein the genetic alteration is a mutation in a gene encoding a RAS protein.  
     
     
         26 . The method of  claim 25 , wherein the genetic alteration is an activated RAS2 va119  gene of  S. cerevisiae.    
     
     
         27 . The method of  claim 23  wherein the genetically altered yeast cells are genetically altered  S. cerevisiae  or genetically altered  S. pombe.    
     
     
         28 . The method of  claim 27  wherein the phenotypic alteration associated with a genetic alteration is selected from the group consisting of: heat shock sensitivity, nitrogen starvation, failure to synthesize normal amounts of glycogen, failure to grow on acetate, failure to mate and failure to sporulate.  
     
     
         29 . The method of  claim 24  wherein the genetic alteration is a disruption of the PDE1 and a disruption of the PDE2 genes of  S. cerevisiae.    
     
     
         30 . The method of  claim 25  wherein the genetic alteration is a disruption of the ras1 gene of  S. pombe  or an activated allele of the ras1 gene of  S. pombe.    
     
     
         31 . A method of identifying a chemical agent which alters activity of a protein encoded by a mammalian gene which, when expressed in genetically altered yeast cells, modifies a phenotypic alteration associated with a genetic alteration in the yeast cells, comprising the steps of: 
 a) expressing the protein encoded by the mammalian gene in genetically altered yeast cells in which the protein encoded by the mammalian gene is not expressed, thereby modifying the phenotypic alteration associated with the genetic alteration;    b) obtaining from genetically altered yeast cells produced in step (a) protein encoded by the mammalian gene;    c) combining protein encoded by the mammalian gene with a chemical agent to be assayed for its ability to alter activity of the protein encoded by the mammalian gene; and    d) determining activity of the protein encoded by the mammalian gene in combination with the chemical agent.