FIG. 6.

The association of Candida Est3p with active telomerase is Est1p dependent. (A) (Top) Extracts were prepared from a set of strains with or without protein A-tagged EST3 (EST3-proA). Total RNAs were isolated from the extracts and subjected to RT-PCR using primers designed to amplify a 350-bp fragment of TER. (Bottom) The same extracts were subjected to IgG-Sepharose pulldown followed by Western blot analysis using antibodies directed against protein A. (B) (Top) Extracts from the indicated strains were subjected to IgG-Sepharose pulldown. Total RNAs on the beads were isolated by proteinase K digestion and ethanol precipitation, followed by RT-PCR to detect TER. (Bottom) Est3-proA-associated telomerases from the indicated strains were isolated by IgG-Sepharose pulldown, followed by direct primer extension analysis using P20 as the primer. (C) (Top) Extracts from the indicated strains were subjected to IgG-Sepharose pulldown. Total RNAs on the beads were isolated by proteinase K digestion and ethanol precipitation, followed by RT-PCR to detect TER. To facilitate quantitative comparison, the BWP17 EST3-proA sample was subjected to threefold serial dilutions prior to RT-PCR. Thermocycling was performed for 20 or 23 cycles to ensure the linearity of the signals. est1-ΔΔ, est1Δ/est1Δ; est3-ΔΔ, est3Δ/est3Δ.