Reporter assay screens for protein targets in saccharomyces cerevisiae

Abstract

The present invention comprises responsive promoters along with screening methods which make use of the promoters in order to identify anti-fungal substances.

1 . An isolated hybrid polynucleotide comprising a member selected from the group consisting of:
   S. cerevisiae  YOR387C Promoter,  S. cerevisiae  YMR175W Promoter,  S. cerevisiae  YFR026C Promoter,  S. cerevisiae  YJL153C Promoter,  S. cerevisiae  YPL033C Promoter,  S. cerevisiae  YOL058W Promoter,  S. cerevisiae  YOR255W Promoter,  S. cerevisiae  YDR250C Promoter,  S. cerevisiae  YDR446W Promoter,  S. cerevisiae  YDR536W Promoter,  S. cerevisiae  YOR255W Promoter,  S. cerevisiae  YDL243C Promoter  S. cerevisiae  YDR256C Promoter,  S. cerevisiae  YFL020C Promoter,  S. cerevisiae  YPL205C Promoter,  S. cerevisiae  YGL205W Promoter,  S. cerevisiae  YGL117W Promoter,  S. cerevisiae  YHR029C Promoter,  S. cerevisiae  YML116W Promoter  S. cerevisiae  YJR109C Promoter,  S. cerevisiae  YCL030C Promoter,  S. cerevisiae  YLR338W Promoter,  S. cerevisiae  YIL066W-A Promoter,  S. cerevisiae  YNL093W Promoter,  S. cerevisiae  YLR381W Promoter,  S. cerevisiae  YKL159C Promoter,  S. cerevisiae  YIL058W Promoter,  S. cerevisiae  YKR037C Promoter,  S. cerevisiae  YNL279W Promoter,  S. cerevisiae  YOR032C Promoter,  S. cerevisiae  YML058W Promoter,  S. cerevisiae  YMR303C Promoter,  S. cerevisiae  YJL153C Promoter  S. cerevisiae  YLR092W Promoter,  S. cerevisiae  YFL052W Promoter,  S. cerevisiae  YIR017C Promoter,  S. cerevisiae  YLL062C Promoter,  S. cerevisiae  YMR323W Promoter  S. cerevisiae  YPL171C Promoter,  S. cerevisiae  YER065C Promoter,  S. cerevisiae  YDR114C Promoter,  S. cerevisiae  YHR082C Promoter,  S. cerevisiae  YDL126C Promoter  S. cerevisiae  YBR106W Promoter,  S. cerevisiae  YLL043W Promoter,  S. cerevisiae  YPR108W Promoter,  S. cerevisiae  YIL041W Promoter,  S. cerevisiae  YJL166W Promoter  S. cerevisiae  YDR165W Promoter,  S. cerevisiae  YLR330 W Promoter,  S. cerevisiae  YDL058W Promoter,  S. cerevisiae  YHR012W Promoter,  S. cerevisiae  YKL053W Promoter,  S. cerevisiae  YKL196C Promoter,  S. cerevisiae  YDL103C Promoter  S. cerevisiae  YLR427W Promoter,  S. cerevisiae  YLL020C Promoter and  S. cerevisiae  YBL065W Promoter; operably linked to a reporter.   
     
     
         2 . An isolated polynucleotide of  claim 1  wherein said promoter comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-41 and 52-68; and/or the reporter comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 42-51. 
     
     
         3 . An isolated vector comprising a polynucleotide of  claim 1 . 
     
     
         4 . An isolated host cell comprising a vector of  claim 3 . 
     
     
         5 . An isolated plasmid polynucleotide comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 85-88. 
     
     
         6 . A method for identifying a substance that inhibits fungal cell growth comprising:
 (a) introducing a hybrid comprising a promoter selected from the group consisting of  S. cerevisiae  YOR387C Promoter,  S. cerevisiae  YMR175W Promoter,  S. cerevisiae  YFR026C Promoter,  S. cerevisiae  YJL153C Promoter,  S. cerevisiae  YPL033C Promoter,  S. cerevisiae  YOL058W Promoter,  S. cerevisiae  YOR255W Promoter,  S. cerevisiae  YDR250C Promoter,  S. cerevisiae  YDR446W Promoter,  S. cerevisiae  YDR536W Promoter,  S. cerevisiae  YOR255W Promoter,  S. cerevisiae  YDL243C Promoter  S. cerevisiae  YDR256C Promoter,  S. cerevisiae  YFL020C Promoter,  S. cerevisiae  YPL205C Promoter,  S. cerevisiae  YGL205W Promoter,  S. cerevisiae  YGL117W Promoter,  S. cerevisiae  YHR029C Promoter,  S. cerevisiae  YML116W Promoter  S. cerevisiae  YJR109C Promoter,  S. cerevisiae  YCL030C Promoter,  S. cerevisiae  YLR338W Promoter,  S. cerevisiae  YIL066W-A Promoter,  S. cerevisiae  YNL093W Promoter,  S. cerevisiae  YLR381W Promoter,  S. cerevisiae  YKL159C Promoter,  S. cerevisiae  YIL058W Promoter,  S. cerevisiae  YKR037C Promoter,  S. cerevisiae  YNL279W Promoter,  S. cerevisiae  YOR032C Promoter,  S. cerevisiae  YML058W Promoter,  S. cerevisiae  YMR303C Promoter,  S. cerevisiae  YJL153C Promoter  S. cerevisiae  YLR092W Promoter,  S. cerevisiae  YFL052W Promoter,  S. cerevisiae  YIR017C Promoter,  S. cerevisiae  YLL062C Promoter,  S. cerevisiae  YMR323W Promoter,  S. cerevisiae  YPL171C Promoter,  S. cerevisiae  YER065C Promoter and  S. cerevisiae  YDR114C Promoter; operably linked to a reporter; into a suitable fungal cell;   (b) contacting the cell with a substance to be tested for the ability to inhibit fungal cell growth; and   (c) detecting expression driven by the promoter in the hybrid;   wherein the substance is identified as a fungal growth inhibitor if the substance modulates expression from the promoter.   
     
     
         7 . A method of  claim 6  wherein the promoter comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-41 and 68. 
     
     
         8 . A method of  claim 6  further comprising a negative-control comprising:
 (i) introducing the hybrid into a suitable fungal cell; 
 (ii) contacting the cell with a blank substance which is known to not modulate expression from the promoter in the hybrid; and 
 (iii) detecting expression driven by the promoter in the hybrid; and 
 (iv) comparing the expression level from the promoter in the cell contacted with the blank substance with the expression level from the promoter in the cell contacted with the substance to be tested for the ability to inhibit fungal cell growth. 
 
     
     
         9 . A method of  claim 6  further comprising a positive-control comprising:
 (i) introducing the hybrid into a suitable fungal cell; 
 (ii) contacting the cell with a positive-control which is known to modulate expression from the promoter in the hybrid; 
 (iii) detecting expression driven by the promoter in the hybrid; and 
 (iv) comparing the expression level from the promoter in the cell contacted with the positive-control substance with the expression level from the promoter in the cell contacted with the substance to be tested for the ability to inhibit fungal cell growth. 
 
     
     
         10 . A method for identifying a substance that inhibits fungal cell growth comprising:
 (a) introducing, into a suitable fungal cell, a first hybrid comprising a first promoter selected from the group consisting of:  S. cerevisiae  YOR387C Promoter,  S. cerevisiae  YMR175W Promoter,  S. cerevisiae  YFR026C Promoter,  S. cerevisiae  YJL153C Promoter,  S. cerevisiae  YPL033C Promoter,  S. cerevisiae  YOL058W Promoter,  S. cerevisiae  YOR255W Promoter,  S. cerevisiae  YDR250C Promoter,  S. cerevisiae  YDR446W Promoter,  S. cerevisiae  YDR536W Promoter,  S. cerevisiae  YOR255W Promoter,  S. cerevisiae  YDL243C Promoter  S. cerevisiae  YDR256C Promoter,  S. cerevisiae  YFL020C Promoter,  S. cerevisiae  YPL205C Promoter,  S. cerevisiae  YGL205W Promoter,  S. cerevisiae  YGL117W Promoter,  S. cerevisiae  YHR029C Promoter,  S. cerevisiae  YML116W Promoter  S. cerevisiae  YJR109C Promoter,  S. cerevisiae  YCL030C Promoter,  S. cerevisiae  YLR338W Promoter,  S. cerevisiae  YIL066W-A Promoter,  S. cerevisiae  YNL093W Promoter,  S. cerevisiae  YLR381W Promoter,  S. cerevisiae  YKL159C Promoter,  S. cerevisiae  YIL058W Promoter,  S. cerevisiae  YKR037C Promoter,  S. cerevisiae  YNL279W Promoter,  S. cerevisiae  YOR032C Promoter,  S. cerevisiae  YML058W Promoter,  S. cerevisiae  YMR303C Promoter,  S. cerevisiae  YJL153C Promoter  S. cerevisiae  YLR092W Promoter,  S. cerevisiae  YFL052W Promoter,  S. cerevisiae  YIR017C Promoter,  S. cerevisiae  YLL062C Promoter,  S. cerevisiae  YMR323W Promoter  S. cerevisiae  YPL171C Promoter,  S. cerevisiae  YER065C Promoter,  S. cerevisiae  YDR114C Promoter and  S. cerevisiae  YBL065W Promoter; operably linked to a first reporter;   and a second hybrid comprising a second promoter selected from the group consisting of:  S. cerevisiae  YHR082C Promoter,  S. cerevisiae  YDL126C Promoter  S. cerevisiae  YBR106W Promoter,  S. cerevisiae  YLL043W Promoter,  S. cerevisiae  YPR108W Promoter,  S. cerevisiae  YIL041W Promoter,  S. cerevisiae  YJL166W Promoter  S. cerevisiae  YDR165W Promoter,  S. cerevisiae  YLR330 W Promoter,  S. cerevisiae  YDL058W Promoter,  S. cerevisiae  YHR012W Promoter,  S. cerevisiae  YKL053W Promoter,  S. cerevisiae  YKL196C Promoter,  S. cerevisiae  YDL103C Promoter  S. cerevisiae  YLR427W Promoter and  S. cerevisiae  YLL020C Promoter; operably linked to a second reporter; wherein said first and second reporters are different;   (b) contacting the cell with a substance to be tested for the ability to inhibit fungal cell growth; and   (c) detecting expression driven from the first and second promoters;   wherein the substance is identified as a fungal growth inhibitor if the substance causes the first promoter expression level to increase or decrease in relation to the expression level from the second reporter.   
     
     
         11 . The method of  claim 10  further comprising a negative-control comprising:
 (i) introducing said first and second hybrids into a suitable fungal cell; 
 (ii) contacting the cell with a blank substance which is known to not modulate expression from the promoters in the hybrids; 
 (iii) detecting expression driven by the promoters in the hybrids; and 
 (iv) comparing the expression level from the first promoter in relation to the expression level from the second promoter in the cell contacted with the blank substance with the expression level from the first promoter in relation to the expression level from the second promoter in the cell contacted with the substance to be tested for the ability to inhibit fungal cell growth. 
 
     
     
         12 . The method of  claim 10  further comprising a positive-control comprising:
 (i) introducing said first and second hybrids into a suitable fungal cell; 
 (ii) contacting the cell with a positive-control substance which is known to modulate expression from the promoters in the hybrids; 
 (iii) detecting expression driven by the promoters in the hybrids; and 
 (iv) comparing the expression level from the first promoter in relation to the expression level from the second promoter in the cell contacted with the positive-control substance with the expression level from the first promoter in relation to the expression level from the second promoter in the cell contacted with the substance to be tested for the ability to inhibit fungal cell growth. 
 
     
     
         13 . A method of  claim 10  wherein the first promoter comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-41 and 68; the second promoter comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 52-67; and/or the reporter comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 42-51. 
     
     
         14 . A method for determining whether a substance inhibits growth of a fungal cell comprising:
 (a) introducing a hybrid comprising a promoter selected from the group consisting of:  S. cerevisiae  YHR082C Promoter,  S. cerevisiae  YDL126C Promoter  S. cerevisiae  YBR106W Promoter,  S. cerevisiae  YLL043W Promoter,  S. cerevisiae  YPR108W Promoter,  S. cerevisiae  YIL041W Promoter,  S. cerevisiae  YJL166W Promoter  S. cerevisiae  YDR165W Promoter,  S. cerevisiae  YLR330 W Promoter,  S. cerevisiae  YDL058W Promoter,  S. cerevisiae  YHR012W Promoter,  S. cerevisiae  YKL053W Promoter,  S. cerevisiae  YKL196C Promoter,  S. cerevisiae  YDL103C Promoter  S. cerevisiae  YLR427W Promoter and  S. cerevisiae  YLL020C Promoter; operably linked to a reporter into a suitable fungal cell;   (b) contacting the cell with a substance to be tested for the ability to inhibit growth of the cell; and   (c) detecting signal from the reporter;   wherein the substance is identified as a fungal growth inhibitor if the substance causes a reduction in the signal from the reporter.   
     
     
         15 . The method of  claim 14  further comprising a negative-control comprising:
 (i) introducing the hybrid into a suitable fungal cell; 
 (ii) contacting the cell with a blank substance which is known to not inhibit fungal cell growth; 
 (iii) detecting signal from the reporter in the hybrid; and 
 (iv) comparing the signal from the reporter in the cell contacted with the blank substance with the signal from the reporter in the cell contacted with the substance to be tested for the ability to inhibit fungal cell growth. 
 
     
     
         16 . The method of  claim 14  further comprising a positive-control comprising:
 (i) introducing the hybrid into a suitable fungal cell; 
 (ii) contacting the cell with a positive-control substance which is known to inhibit fungal cell growth; 
 (iii) detecting signal from the reporter in the hybrid; and 
 (iv) comparing the signal from the reporter in the cell contacted with the positive-control substance with the signal from the reporter in the cell contacted with the substance to be tested for the ability to inhibit fungal cell growth. 
 
     
     
         17 . A method of  claim 14  wherein the promoter comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 52-67; and/or the reporter comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 42-51. 
     
     
         18 . A method for determining whether a substance inhibits growth of a fungal cell comprising:
 (a) introducing a first hybrid comprising a first promoter selected from the group consisting of:     S. cerevisiae  YHR082C Promoter,  S. cerevisiae  YDL126C Promoter  S. cerevisiae  YBR106W Promoter,  S. cerevisiae  YLL043W Promoter,  S. cerevisiae  YPR108W Promoter,  S. cerevisiae  YIL041W Promoter,  S. cerevisiae  YJL166W Promoter  S. cerevisiae  YDR165W Promoter,  S. cerevisiae  YLR330 W Promoter,  S. cerevisiae  YDL058W Promoter,  S. cerevisiae  YHR012W Promoter,  S. cerevisiae  YKL053W Promoter,  S. cerevisiae  YKL196C Promoter,  S. cerevisiae  YDL103C Promoter  S. cerevisiae  YLR427W Promoter and  S. cerevisiae  YLL020C Promoter; operably linked to a first reporter;   and a second hybrid comprising a second promoter selected from the group consisting of:     S. cerevisiae  YHR082C Promoter,  S. cerevisiae  YDL126C Promoter  S. cerevisiae  YBR106W Promoter,  S. cerevisiae  YLL043W Promoter,  S. cerevisiae  YPR108W Promoter,  S. cerevisiae  YIL041W Promoter,  S. cerevisiae  YJL166W Promoter  S. cerevisiae  YDR165W Promoter,  S. cerevisiae  YLR330 W Promoter,  S. cerevisiae  YDL058W Promoter,  S. cerevisiae  YHR012W Promoter,  S. cerevisiae  YKL053W Promoter,  S. cerevisiae  YKL196C Promoter,  S. cerevisiae  YDL103C Promoter  S. cerevisiae  YLR427W Promoter and  S. cerevisiae  YLL020C Promoter; operably linked to a second reporter into a suitable fungal cell; wherein said first and second promoter are different and wherein said first and second reporter are different;   (b) contacting the cell with a substance to be tested for the ability to inhibit growth of the cell; and   (c) detecting signal from the reporters;   wherein the substance is identified as a fungal growth inhibitor if the substance causes a reduction in the signal from both the first and second reporters.   
     
     
         19 . The method of  claim 18  further comprising a negative-control comprising:
 (i) introducing the first and second hybrids into a suitable fungal cell; 
 (ii) contacting the cell with a blank substance which is known to not inhibit fungal cell growth; 
 (iii) detecting signal from first and second reporters in the hybrids; and 
 (iv) comparing the signal from the first and second reporters in the cell contacted with the blank substance with the signal from the first and second reporters in the cell contacted with the substance to be tested for the ability to inhibit fungal cell growth. 
 
     
     
         20 . The method of  claim 18  further comprising a positive-control comprising:
 (i) introducing the first and second hybrids into a suitable fungal cell; 
 (ii) contacting the cell with a positive control substance which is known to inhibit fungal cell growth; 
 (iii) detecting signal from first and second reporters in the hybrids; and 
 (iv) comparing the signal from the first and second reporters in the cell contacted with the positive control substance with the signal from the first and second reporters in the cell contacted with the substance to be tested for the ability to inhibit fungal cell growth. 
 
     
     
         21 . A method of  claim 18  wherein said promoters comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs: 52-67; and/or the reporter comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 42-51. 
     
     
         22 . A method for determining whether a substance inhibits growth of a malignant cell comprising
 (a) introducing, into a suitable fungal cell, a first hybrid comprising a first promoter selected from the group consisting of:  S. cerevisiae  YLR338W,  S. cerevisiae  YIL066W,  S. cerevisiae  YNL093W,  S. cerevisiae  YDR250C,  S. cerevisiae  YLR381W,  S. cerevisiae  YBL065W,  S. cerevisiae  YMR323W and  S. cerevisiae  YPL171C; operably linked to a first reporter; and   a second hybrid comprising a second promoter selected from the group consisting of:     S. cerevisiae  YHR082C Promoter,  S. cerevisiae  YDL126C Promoter  S. cerevisiae  YBR106W Promoter,  S. cerevisiae  YLL043W Promoter,  S. cerevisiae  YPR108W Promoter,  S. cerevisiae  YIL041W Promoter,  S. cerevisiae  YJL166W Promoter  S. cerevisiae  YDR165W Promoter,  S. cerevisiae  YLR330 W Promoter,  S. cerevisiae  YDL058W Promoter,  S. cerevisiae  YHR012W Promoter,  S. cerevisiae  YKL053W Promoter,  S. cerevisiae  YKL196C Promoter,  S. cerevisiae  YDL103C Promoter  S. cerevisiae  YLR427W Promoter and  S. cerevisiae  YLL020C Promoter; operably linked to a second reporter;   (b) contacting the cell with a substance to be tested for the ability to inhibit growth of a malignant cell; and   (c) detecting expression driven by the first and second promoters;   wherein the substance is identified as a malignant cell growth inhibitor if the substance causes the expression level from the first promoter to increase or decrease in relation to the expression level from the second promoter.   
     
     
         23 . The method of  claim 22  wherein the first promoter comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 8, 22-25, 38, 39 and 68; and/or the second promoter comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 52-67. 
     
     
         24 . The method of  claim 22  further comprising a negative-control comprising:
 (i) introducing said first and second hybrids into a suitable fungal cell; 
 (ii) contacting the cell with a blank substance which is known to not modulate expression from the promoters in the hybrids; and 
 (iii) detecting expression driven by the promoters in the hybrids; 
 (iv) comparing the expression level from the first promoter in realtion to the expression level from the second promoter in the cell contacted with the blank substance with the expression level from the first promoter in realtion to the expression level from the second promoter in the cell contacted with the substance to be tested for the ability to inhibit fungal cell growth. 
 
     
     
         25 . The method of  claim 22  further comprising a positive-control comprising:
 (i) introducing said first and second hybrids into a suitable fungal cell; 
 (ii) contacting the cell with a positive-control substance which is known to modulate expression from the promoters in the hybrids; 
 (iii) detecting expression driven by the promoters in the hybrids; and 
 (iv) comparing the expression level from the first promoter in realtion to the expression level from the second promoter in the cell contacted with the positive control substance with the expression level from the first promoter in realtion to the expression level from the second promoter in the cell contacted with the substance to be tested for the ability to inhibit fungal cell growth.