QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

Help viewing high resolution images Return to article

Fig. 5. Rescue of Skp1 hydroxylation and culmination in mutant cells by P4H1 or PKAcat cDNAs. (A) Ax3, bsr⁺ (+, normal), P4H1^- clones (-) and P4H1^- clones complemented with P4H1-myc under control of its own promoter (P), the pre-stalk-specific ecmA(FL) promoter (E), the pre-spore-specific cotB promoter (C) or the discoidin promoter (D), were developed for 16 hours under ambient conditions and analyzed by western blotting with myc-specific mAb 9E10 for P4H1-myc accumulation. The position of P4H1-myc relative to the more intense background bands, which serve as loading controls, is indicated. (B) Parallel samples were analyzed by western blotting for Skp1 isoforms using mAb 4E1. Markers indicate high (glycosylated) and low M_r (non-hydroxylated/glycosylated) Skp1 isoforms, and a lower band of unknown identity. Results in A and B are typical of three experiments, including independently complemented HW289-derived cells (not shown). (C) Quantitation of culmination under ambient conditions by spore counts. No rescue was observed using an empty vector (not shown). P4H1^- cells transfected with PKAcat (strain HW410) are also shown. (D,E) Complementation with P4H1-mutants. (D) Fifteen-hour slugs from Ax3, or P4H1a^- expressing the P4H1-myc mutants D132N (DNa and DNb are independent clones) or R276A (RA) under control of the ecmA-promoter, were analyzed for P4H1-myc expression as in A, and for Skp1 electrophoretic mobility as in B. (E) Strains were developed at the indicated O₂ level for the indicated time under strong overhead light. P4H1^- cells exhibited breakthrough at 21% O₂ in this trial, but were selectively inhibited relative to Ax3 at 15% O₂. Spore numbers (shown) correlated with morphological appearance (not shown).

Return to article