Figure 1
His6-Rif1-Flag3 protein is functional. (A,B) The wild-type or rif1∆ harboring pREP81 vector, or rif1∆ harboring pREP81-Rif1 or pREP81-His6-Rif1-Flag3 were grown in SD +2 µM thiamine, and cells were washed with EMM without thiamine and transferred to the EMM medium containing the indicated concentration (2 µM or 5 µM) of thiamine for 16 hrs. (A) The expression levels of Rif1 and tubulin were examined by western blotting. (B) Cell cycle was analyzed by FACS. (C) The 5-fold serial dilution of the cells, indicated in the figure, was spotted on EMM plate containing 5 or 15 μM thiamine. The plates were incubated at 25 °C or 30 °C. The photographs were taken at 4 days after plating. (D) Function in telomere length regulation. Genomic DNA was extracted from the cells indicated, digested with EcoRI and electrophoresed on 1.2% agarose in TAE buffer. The digested DNA was transferred to nitrocellulose membrane and fixed with UV irradiation, followed by hybridization with 32P-labeled telomere DNA. Left, EtBr-stained gel; right, Southern blotting of the same gel.
