Activation induced deaminase (aid)

Abstract

The invention is directed to a cell comprising a nucleic acid encoding an Activation Induced Deaminase (AID) polypeptide, a fusion protein comprising an AID polypeptide, and methods of using a nucleic acid encoding an AID polypeptide.

1 . A cell comprising a nucleic acid encoding an Activation Induced Deaminase (AID) polypeptide, or an AID variant, derivative or homologue, and having a mutator phenotype. 
     
     
         2 . The cell of  claim 1 , wherein said cell is a prokaryotic cell. 
     
     
         3 . The cell of  claim 1 , wherein said cell is an eukaryotic cell. 
     
     
         4 . The cell of  claim 1 , wherein the AID homologue is Apo1bec-1, Apobec-1, Apobec3C or Apobec3G. 
     
     
         5 . A fusion protein comprising an AID polypeptide, or AID variant, derivative or homologue thereof, having a mutator phenotype operably linked to one half of a specific binding pair. 
     
     
         6 . The fusion protein of  claim 5 , wherein said one half of a specific binding pair is a DNA binding domain. 
     
     
         7 . A vector comprising a nucleic acid encoding the fusion protein of  claim 5 . 
     
     
         8 . A cell comprising a nucleic acid encoding the fusion protein of  claim 5 . 
     
     
         9 . A method for preparing a gene product having a desired activity, comprising the steps of:
 a) expressing a nucleic acid encoding the gene product in a population of cells according to  claim 1 ;   b) identifying a cell or cells within the population of cells which expresses a mutant gene product having the desired activity; and   c) establishing one or more clonal populations of cells from the cell or cells identified in step (b), and selecting from said clonal populations a cell or cells which expresses a gene product having an improved desired activity.   
     
     
         10 . A method as claimed in  claim 9 , wherein the nucleic acid encoding the gene product is operably linked to the second half of a specific binding pair. 
     
     
         11 . A method of directing mutation to a specific gene product of interest comprising:
 i) generating a nucleic acid construct comprising a nucleic acid sequence encoding a specific gene product operably linked to a DNA binding protein recognition sequence;   ii) transfecting said nucleic acid construct into a population of host cells expressing the fusion protein of  claim 6 ;   ii) incubating said transfected host cells under conditions suitable for allowing the specific binding pairing of DNA binding protein to DNA binding protein recognition sequence to occur;   iv) identifying a cell or cells within the population of cells which expresses a mutant gene product having the desired activity; and   v) establishing one or more clonal populations of cells from the cell or cells   identified in (iv), and selecting from said clonal populations a cell or cells which expresses a gene product having an improved desired activity.   
     
     
         12 . A method of identifying components of AID-dependent mutation activity comprising expressing AID in a cell deficient in expression or activity of a known gene and assessing mutator activity compared to activity in a cell expressing said gene. 
     
     
         13 . A method of screening for a modulator of AID activity comprising:
 (a) expressing AID in a prokaryotic cell;   (b) maintaining the AID-expressing prokaryotic cell in the presence of a selectable medium;   (c) detecting the presence of colonies in the absence or presence of a test compound, wherein a modified number of colonies when compared to a sample in the absence of a test compound is indicative of the ability of the test compound to modify AID mutator activity.   
     
     
         14 . A method of inducing a mutation in a cell comprising administering an AID polypeptide or functional homologue thereof. 
     
     
         15 . A method for treating a disorder characterized by an increased mutation rate, comprising administering an agent that modifies AID functional activity or gene expression. 
     
     
         16 . A method of decreasing hypermutation/resistance to a compound such as an antibiotic in a population of bacteria comprising modulating activity of a bacterial AID homologue. 
     
     
         17 . A construct for use in the method of  claim 11 , said construct comprising a coding sequence for the gene product of interest, wherein said coding sequence is placed under the control of a first promoter upstream of the coding sequence and further comprising a second promoter downstream of the coding sequence, wherein said first and second promoters are arranged in opposing orientation so as to allow convergent transcription of the coding sequence.