METHOD FOR IDENTIFYING CELLULAR GROWTH INHIBITORS

The present invention is a method which permits the selective screening for growth-inhibiting substances having a known mechanism of action; i.e., substances which inhibit or otherwise interfere with an enzyme or other gene product whose function is required for the growth or survival of the cell. The method employs cells (e.g., bacterial cells) that contain a nucleic acid fragment that encodes an RNA fragment that can interfere with the expression of a gene product (e.g., an anti-sense RNA that hybridizes to a mRNA), wherein expression of the RNA fragment pre-sensitizes the cell to substances (drugs) that act at the gene product (e.g., a protein or RNA). The cells lose the capability to express the RNA fragment. In the method, the recombinant cells are grown in a nutrient medium in the presence of a test substance under conditions in which expression of the RNA fragment occurs at a level that pre-sensitizes the cell to substances that act at the targeted gene product. The growth conditions are also controlled such that the cells lose the capability to express the RNA fragment. When the test substance is a growth inhibitor that acts on the targeted gene product, the cells lacking the RNA fragment (revertant cells) will have a growth advantage over cells containing the RNA fragment, and the growth of revertant cells will occur. The method of the invention includes monitoring the cell growth for the appearance of revertant cells, which leads to the identification of selective growth inhibitors having a specific mode of action. La présente invention concerne une méthode qui permet le criblage sélectif de substance inhibitrices de croissance présentant un mécanisme d'action connu; c'est-à-dire, des substances qui inhibent ou différemment interfèrent avec une enzyme ou un autre produit génétique dont la fonction est nécessaire pour la croissance ou pour la survie de la cellule. Ladite méthode fait appel à des cellules (par exemple, des cellules bactériennes) qui contiennent un fragment d'acide nucléique codant pour un fragment d'ARN qui peut interférer avec l'expression d'un produit génétique (par exemple, un ARN antisens qui s'hybride avec un ARNm), l'expression du fragment d'ARN pré-sensibilisant la cellule aux substances (médicaments) qui agissent au niveau du produit génétique (par exemple, une protéine ou un ARN). Les cellules perdent la capacité à exprimer le fragment d'ARN. Selon ladite méthode, les cellules recombinantes sont cultivées dans un milieu nu