Fig. 5.

Evidence that transcription occurs during germination. (A) An in vitro transcription reaction was carried out in whole extracts from vegetative cells of PY79 (WT) as well as from dormant and germinating spores of PY79 (WT), BZ90 (sigA-R365A), and BZ91 (sigA-R365D) strains and in a transcription buffer supplemented with NTPs (ATP, CTP, GTP, UTP, [α-32P]-UTP). After 40 min of incubation at 37 °C, the reaction was stopped, RNA was purified, radioactively labeled RNAs were analyzed in 8% polyacrylamide gels, and bands were visualized by autoradiography. The analysis was carried out in two biological repeats, and a representative experiment is presented. (B) Spores of PY79 (WT), BZ90 (sigA-R365A), and BZ91 (sigA-R365D) strains were incubated with 10 mM l-Ala to trigger germination, collected by centrifugation at the indicated time, and their RNAs were extracted. The mRNA levels of selective genes tig, rpmE, and pupG during germination were determined by quantitative RT-PCR. The result is presented as the fold change of target gene expression after germination relative to before germination. The assays were carried out in triplicate, and representative data are presented. (C) Total RNA was extracted from vegetative cells, dormant and germinating spores of PY79 (WT), BZ90 (sigA-R365A), and BZ91 (sigA-R365D) strains and subjected to Northern blot analysis using rpmE (201 nt) and pupG (2001 nt) specific biotinylated probes. knockout (KO), indicates control extracts derived from vegetative cells of the corresponding LS26 (∆rpmE) and LS76 (∆pupG) KO strains. The analysis was carried out in three independent biological repeats, and a representative experiment is presented. Numbers on the right correspond to RNA size marker.