Figure 5. The TopBP1 transcriptional-activation and -repressor domains function in yeast.

(a) A schematic representation of the Gal4 DBD fusion proteins tested in yeast. (b) The ability of yeast expressing the proteins shown in (a) to activate transcription in yeast was monitored by plating cells out on SC−Trp medium, where they should all grow due to the presence of the plasmid, and SC−Trp−His−Ade medium, where growth should only occur following transcriptional-activation by the Gal4–TopBP1 fusion proteins. Growth was only seen on the triple-dropout medium with the Gal4–TopBP1^253–591 expressing yeast. (c) The ability of the fusion proteins to activate transcription in yeast was tested directly by monitoring levels of β-galactosidase activity. The promoter controlling the expression of this protein is controlled by Gal4 DNA-binding sites. The results are expressed as attenuance units following the β-galactosidase assay. (d) Expression of the fusion proteins in yeast was confirmed by Western blotting using an anti-Gal4 antibody; the numbers to the left-hand side of the blot represent the marker proteins sizes in kDa. The Gal4–TopBP1^2–258 fusion protein major band has a higher molecular mass than predicted. The predicted size is marked by a * where a protein can be detected.