Gene function analysis by amber stop codon suppression: CMBF is a nuclear protein that supports growth and development of Dictyostelium amoebae

The C-module-binding factor, CMBF, is a nuclear DNA-binding protein which w as originally identified through its specific binding to a promoter element within the retrotransposable element TRE5-A of Dictyostelium discoideum AX 2 cells. in order to analyse putative physiological functions of CMBF for t he TRE5-A-hosting D. discoideum cells, we used a novel strategy to create m utant cell lines which stably underexpressed functional CMBF. An amber (UAG ) translation stop codon was introduced into the chromosomal copy of the CM BF-encoding gene (cbfA), and an amber suppressor tRNA gene was expressed in the same mutant cells. Due to the low efficiency of translation stop codon suppression in this system all recovered cell lines expressed <20% of wild -type CMBF levels. The mutant cell lines displayed strong growth phenotypes when plated on their natural food source, bacteria. We show evidence that growth reduction was due to impaired phagocytosis of bacteria in the mutant s. Ail obtained mutants showed a strong developmental defect which was defi ned by the formation of very small fruiting bodies. The strength of the dev elopmental phenotype appeared to depend upon the residual CMBF levels maint ained in the mutants. We propose that CMBF is a general transcription regul ator which supports the normal expression of several genes required for the maintenance of high proliferation rates of D. discoideum amoebae as well a s proper aggregation and development. Our results demonstrate that amber st op codon suppression may be a useful strategy to stably underexpress protei ns whose coding genes cannot be successfully disrupted by homologous recomb ination. (C) 2001 Academic Press.