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 was originally identified through its specific binding to a promoter element within the retrotransposable element TRE5-A of Dictyostelium discoideum AX2 cells. In order to analyse putative physiological functions of CMBF for the TRE5-A-hosting D. discoideum cells, we used a novel strategy to create mutant cell lines which stably underexpressed functional CMBF. An amber (UAG) translation stop codon was introduced into the chromosomal copy of the CMBF-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 mutants. All obtained mutants showed a strong developmental defect which was defined by the formation of very small fruiting bodies. The strength of the developmental phenotype appeared to depend upon the residual CMBF levels maintained in the mutants. We propose that CMBF is a general transcription regulator which supports the normal expression of several genes required for the maintenance of high proliferation rates of D. discoideum amoebae as well as proper aggregation and development. Our results demonstrate that amber stop codon suppression may be a useful strategy to stably underexpress proteins whose coding genes cannot be successfully disrupted by homologous recombination.
Winckler
T
T
Institut für Pharmazeutische Biologie, Universität Frankfurt/M. (Biozentrum), D-60439 Frankfurt am Main, Germany. winckler@em.uni-frankfurt.de
Trautwein
C
C
Tschepke
C
C
Neuhäuser
C
C
Zündorf
I
I
Beck
P
P
Vogel
G
G
Dingermann
T
T
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D000818:Animals; D002455:Cell Division; D018388:Codon, Terminator / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004023:Dictyostelium / Q000166:cytology / Q000235:genetics / Q000254:growth & development* / Q000382:microbiology; D015870:Gene Expression / Q000235:genetics; D018507:Gene Expression Regulation, Developmental; D016153:Genes, Suppressor / Q000235:genetics; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010587:Phagocytosis; D010641:Phenotype; D010873:Pinocytosis; D014176:Protein Biosynthesis; D015800:Protozoan Proteins; D013489:Suppression, Genetic / Q000235:genetics*
D018388:Codon, Terminator; D004268:DNA-Binding Proteins; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D015800:Protozoan Proteins; C400205:cbfA protein, Dictyostelium discoideum