Methods for isolating novel antimicrobial agents from hypermutable mammalian cells

Abstract

Dominant-negative alleles of human mismatch repair genes can be used to generate hypermutable cells and organisms. By introducing these genes into mammalian cells new cell lines with novel and useful properties can be prepared more efficiently than by relying on the natural rate of mutation or introduction of mutations by chemical mutagens. These methods are useful for generating novel and highly active antimicrobial molecules as well as superior antimicrobial agents from pre-existing chemicals. These methods are also useful for generating cell lines expressing novel antimicrobials that are useful for pharmaceutical manufacturing.

What is claimed is:  
     
         1 . A method for making a mammalian cell hypermutable, comprising: 
 introducing into said mammalian cell a polynucleotide comprising a dominant-negative allele of a mismatch repair gene, whereby said cell becomes hypermutable.    
     
     
         2 . The method of  claim 1  wherein said polynucleotide is introduced into said cell by transfection in vitro.  
     
     
         3 . The method of  claim 1 , wherein said polynucleotide is introduced into said cell by transfection of an adherent cell in vitro.  
     
     
         4 . The method of  claim 1  wherein said mismatch repair gene is PMS2.  
     
     
         5 . The method of  claim 1  wherein said mismatch repair gene is human PMS2.  
     
     
         6 . The method of  claim 1  wherein said mismatch repair gene is human MLH1.  
     
     
         7 . The method of  claim 1  wherein said mismatch repair gene is human PMS1.  
     
     
         8 . The method of  claim 1  wherein said mismatch repair gene is human MSH2.  
     
     
         9 . The method of  claim 4  wherein said allele comprises a truncation mutation.  
     
     
         10 . The method of  claim 4  wherein said allele comprises a truncation mutation at codon 134.  
     
     
         11 . The method of  claim 9  wherein said truncation mutation is a thymidine at nucleotide 424 of wild-type PMS2.  
     
     
         12 . A homogeneous composition comprising a cultured, hypermutable, mammalian cell comprising a dominant negative allele of a mismatch repair gene.  
     
     
         13 . The composition of  claim 12  wherein said mismatch repair gene is PMS2.  
     
     
         14 . The composition of  claim 12  wherein said mismatch repair gene is human PMS2.  
     
     
         15 . The composition of  claim 12  wherein said mismatch repair gene is human MLH1.  
     
     
         16 . The composition of  claim 12  wherein said mismatch repair gene is human PMS1.  
     
     
         17 . The composition of  claim 12  wherein said mismatch repair gene is human MSH2.  
     
     
         18 . The composition of  claim 12  wherein said cell expresses a protein consisting of the first 133 amino acids of hPMS2.  
     
     
         19 . A method for obtaining a mammalian cell that is resistant to a selected microbe comprising: 
 growing a culture of mammalian cells wherein said cells have a dominant-negative allele of a mismatch repair gene;    exposing said cells to said selected microbe; and    selecting said mammalian cell that is resistant to said selected microbe.    
     
     
         20 . The method of  claim 19  wherein said hypermutable cell is selected for resistance to a gram-negative microbe.  
     
     
         21 . The method of  claim 19  wherein said hypermutable cell is selected for resistance to a gram-positive microbe.  
     
     
         22 . The method of  claim 19  wherein said hypermutable cell is selected for resistance to a protozoan.  
     
     
         23 . The method of  claim 19  wherein said hypermutable cell is selected for resistance to a bacteria.  
     
     
         24 . The method of  claim 19  wherein said hypermutable cell is selected for resistance to a fungi.  
     
     
         25 . The method of  claim 19  wherein said step of selecting for microbial resistance comprises isolating and testing conditioned medium from said hypermutable cell.  
     
     
         26 . A method for obtaining a cell comprising a mutation in a gene encoding an antimicrobial activity comprising: 
 growing a culture of mammalian cells having said gene encoding said antimicrobial activity, and a dominant negative allele of a mismatch repair gene;    selecting a cell comprising said antimicrobial activity; and    determining whether said gene comprises a mutation.    
     
     
         27 . The method of  claim 26  wherein said hypermutable cell is selected for resistance to a gram-negative microbe.  
     
     
         28 . The method of  claim 26  wherein said hypermutable cell is selected for resistance to a gram-positive microbe.  
     
     
         29 . The method of  claim 26  wherein said hypermutable cell is selected for resistance to a protozoan.  
     
     
         30 . The method of  claim 26  wherein said hypermutable cell is selected for resistance to a bacteria.  
     
     
         31 . The method of  claim 26  wherein said hypermutable cell is selected for resistance to a fungi.  
     
     
         32 . The method of  claim 26  wherein said step of selecting a cell for antimicrobial activity comprises isolating and testing conditioned medium from said hypermutable cell.  
     
     
         33 . The method of  claim 26  wherein said step of examining said cell to determine whether said gene comprises a mutation comprises analyzing a nucleotide sequence of said gene.  
     
     
         34 . The method of  claim 26  wherein said step of examining said cell to determine whether said gene comprises a mutation comprises analyzing mRNA transcribed from said gene.  
     
     
         35 . The method of  claim 26  wherein said step of examining said cell to determine whether said gene comprises a mutation comprises analyzing a protein encoded by said gene.  
     
     
         36 . The method of  claim 26  wherein said step of examining said cell to determine whether said gene comprises a mutation comprises analyzing the phenotype of said gene.