FIGURE 2.

The Asf1-30 mutant protein was unstable at the restrictive temperature. A, the mRNA level of asf1 as monitored by RT-PCR was unchanged between wild type and the asf1-30 mutant. Samples were obtained from wild type and the asf1-30 mutant as indicated. Experiments were done with reverse transcriptase (top two panels) and without reverse transcriptase (bottom two panels). The act1 gene was used as a control. PCR was done through three different cycles (20, 22, and 25). B, instability of the Asf1-30-13Myc protein. The stability of the Asf1 protein was examined at 26 or 36 °C for 0–3 h in the presence of CHX. Wild type and asf1-30 mutant strains carrying the Asf1 protein tagged with the 13Myc epitope on the C terminus were incubated with 100 μg/ml CHX. Asf1 was detected by immunoblotting with an anti-Myc antibody (top). Tubulin was used as a loading control (bottom). C, Asf1-30 was still unstable even in the presence of wild-type Asf1. The asf1⁺-13myc and asf1-30-13myc genes were placed under the inducible nmt1 promoter in pREP41. These genes were first expressed in the asf1-30-GFP mutant strains at 26 °C in the absence of thiamine. Then these cells were shifted up to 36 °C for 0–3 h in the presence of 2 μm thiamine and 100 μg/ml CHX. Both wild-type Asf1-13Myc and mutant Asf1-30-13Myc proteins were detected by immunoblotting with an anti-Myc antibody (top). The Asf1-GFP protein was detected by immunoblotting with an anti-GFP antibody (middle). Tubulin was used as a loading control (bottom).