MGAL: A GAL Gene Switch-Based Suite of Methods for Protein Analyses and Protein Expression in Multicellular Organisms and Cells Therefrom

Abstract

The invention provides methods for detecting and analyzing protein-protein interactions and agonists and antagonists thereof, detecting and analyzing protein sequences, and regulatable gene expression in multicellular organisms or cells therefrom.

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         50 . A galactose-independent method of activating GAL4 protein regulatable gene expression in multicellular organisms or cells therefrom comprising:
 a. introducing a gene expressing a first protein or fragment thereof fused with the amino- or carboxyl-terminus of Gal80p,   b. introducing a gene expressing a second protein or fragment thereof fused to a membrane targeting sequence, and   c. treating the multicellular organism or cell therefrom with an induction agent, wherein treatment mediates the cytoplasmic interaction of the first and second proteins, relieving Gal80p inhibition and permitting Gal4p activation of GAL4 protein regulatable, UASGAL-containing promoters,   wherein the promoters are fused to a heterologous gene.   
     
     
         51 . The method of  claim 50 , wherein Gal4p or a fragment thereof is expressed from a chromosomal Gal4p gene or a heterologous Gal4p gene introduced into the multicellular organisms or cells therefrom. 
     
     
         52 . The method of  claim 50 , wherein the membrane targeting sequences are a myristoylation sequence, mitochondrial outer membrane targeting sequence, or other membrane targeting/anchoring sequences. 
     
     
         53 . The method of  claim 52  wherein the myristoylation sequence is MGCTVSTQTIGDESDP (SEQ ID NO:1). 
     
     
         54 . The method of  claim 52  wherein the mitochondrial outer membrane targeting sequence is the N-terminal sequence of Tom70/Mas70 protein, MKSFITRNKTAILATVAATGTAIGAYYYY (SEQ ID NO:3). 
     
     
         55 . The method of  claim 50 , wherein the first protein is any protein encoded by a cDNA. 
     
     
         56 . The method of  claim 55 , wherein the first protein is Fpri. 
     
     
         57 . The method of  claim 50 , wherein the second protein is any protein encoded by a cDNA. 
     
     
         58 . The method of  claim 57 , wherein the second protein is Cnal. 
     
     
         59 . The method of  claim 50 , wherein the induction agent is any molecule that mediates the interaction of said first and second proteins. 
     
     
         60 . The method of  claim 50 , wherein the induction agent is FK.506. 
     
     
         61 . The method of  claim 50 , wherein the GAL4 protein regulatable genes are expressed from UAS GAL  containing promoters. 
     
     
         62 . The method of  claim 50 , wherein the GAL4 protein regulatable gene is transcribed through any promoter under the control of UAS GAL  sequences and the UASGAL-specific transcriptional activator sequences. 
     
     
         63 . A method of regulatable gene expression in multicellular organisms or cells therefrom comprising:
 a. introducing a recombinant expression construct encoding Gal80p into the multicellular organism or cell therefrom,   b. introducing a recombinant expression construct encoding Gal3p into the multicellular organism or cell therefrom,   c. introducing a recombinant expression construct encoding Gal4p into the multicellular organism or cell therefrom,   d. introducing a coding sequence operably linked to a promoter sensitive to or regulated by the proteins encoded by the expression constructs of steps a-c,   e. treating the multicellular organism or cell therefrom with an inducing agent, wherein treatment mediates the cytoplasmic interaction of the Gal3p and Gal80p, relieving Gal80p inhibition and permitting Gal4p activation of the coding sequence operably linked to the promoter sensitive to or regulated by the Gal80p, Gal3p and Gal4p encoded by the expression constructs of steps a-c,   wherein the promoters are fused to a heterologous gene.   
     
     
         64 . The method of  claim 63 , wherein Gal4p or a fragment thereof is expressed from a heterologous Gal4p gene introduced into the multicellular organisms or cells therefrom. 
     
     
         65 . The method of  claim 63 , wherein the inducing agent is galactose. 
     
     
         66 . The method of  claim 63 , wherein the coding sequence operably linked to a promoter sensitive to or regulated by the proteins encoded by the expression constructs of steps a-c are expressed from UAS GAL -containing promoters. 
     
     
         67 . A method of regulatable gene expression in multicellular organisms or cells therefrom comprising:
 a. introducing a recombinant expression construct encoding Gal80p into the multicellular organism or cell therefrom,   b. introducing a recombinant expression construct encoding Gal3p into the multicellular organism or cell therefrom,   c. introducing a recombinant expression construct encoding Gal4p or a fragment thereof fused to a transcription factor or DNA-binding portion thereof into the multicellular organism or cell therefrom,   d. introducing a coding sequence operably linked to a promoter sensitive to or regulated by the transcription factor,   e. treating the multicellular organism or cell therefrom with an inducing agent, wherein treatment mediates the cytoplasmic interaction of the Gal3p and Gal80p, relieving Gal80p inhibition and permitting Gal4p activation of the coding sequence operably linked to the promoter sensitive to or regulated by the transcription factor.   
     
     
         68 . The method of  claim 67 , wherein Gal4p or a fragment thereof is expressed from a heterologous Gal4p gene introduced into the multicellular organisms or cells therefrom. 
     
     
         69 . The method of  claim 67 , wherein the inducing agent is galactose. 
     
     
         70 . The method of  claim 67 , wherein the promoter that is sensitive to or regulated by the transcription factor is an endogenous promoter. 
     
     
         71 . The method of  claim 67 , wherein the coding sequence operatively linked to the promoter that is sensitive to or regulated by the transcription factor is an endogenous coding sequence. 
     
     
         72 . The method of  claim 67 , wherein the promoter that is sensitive to or regulated by the transcription factor is a heterologous promoter. 
     
     
         73 . The method of  claim 67 , wherein the coding sequence operatively linked to the promoter that is sensitive to or regulated by the transcription factor is a heterologous coding sequence. 
     
     
         74 . The method of  claim 67 , wherein the transcription fragment is a transcription factor from a cell of a multicellular organism.