Figure 9.

Production of Hac1p only from mature mRNA. At the top, the nucleotide sequence of the wild-type (WT) HAC1 gene around the 5′ splice site and its deduced amino acid sequence are shown. In a mutant HAC1 gene designated WT(XbaI), a XbaI site (underlined) was created at nt 640 between the two MfeI sites in YCp-HAC1WT that changed aa 214 and 215 from Leu-Asp to Ser-Arg. Δintron(XbaI) contains a XbaI site in YCp-HAC1Δintron similarly. The Ala221Stop mutant contains a stop codon TAG (double underlined) instead of the codon for Ala²²¹. The Ala221Stop′ mutant contains a 2-nt (TA) insertion between the codons for Pro²²⁰ and Ala²²¹ that creates a stop codon (double underlined) immediately after Pro²²⁰ but maintains nucleotide sequences at the 5′ splice site from the position −2 (AGCCG—). The Asn216Stop mutant contains a stop codon TAG (double underlined) instead of the codon for Asn²¹⁶. The hac1Δ strain (KMY1145) was transformed with a vector alone (V) or each of these constructs as indicated. Transformants were grown at 30°C in SC(−Ura, Leu) medium to midlogarithmic phase, and aliquots were incubated for 3 h in the presence (hatched bars) or absence (solid bars) of tunicamycin (TM). β-Galactosidase activities in cell extracts were determined, and are presented as the mean ± SD, based on duplicate determinations with three independent transformants. Separate samples taken after 1 h were used for extracting total proteins or total RNAs that were analyzed by immunoblotting using anti-Hac1p antibodies or by Northern blot hybridization using DNA probes specific for HAC1 and ACT1. Hac1p of 238 and 220 aa were also translated in vitro. * denotes a nonspecific band.