Figure 2

Determination of transcription start sites of C. merolae rDNA and 5S rDNA. (A) Positions of probes (a–f) used in northern blot analysis for rDNA. (B) Mapping of TSP of rDNA by northern blot analysis. C. merolae total RNA (6 g) prepared from logarithmic-growth cells (approximately OD₇₅₀=0.5) under continuous light conditions was subjected to northern blot analysis with relevant specific probes shown in panel (A). Transcript size is indicated at the left in kilonucleotides. Putative positions of prematured full-length rRNA (full length), prematured 18S rRNA (Pre-18S) and matured 18S rRNA (Matured 18S) are indicated at the right. Lower panel shows rRNA stained with methylene blue as a loading control. (C) 5'-end mapping of rDNA TSP by primer extension and S1 nuclease protection assays. Positions of primer (arrowhead) and DNA probe (line) used in primer extension analysis and S1 nuclease protection assay are indicated at the top. Asterisks indicate ³²P labelled 5'-end of DNA. Primer extension analysis (left) or S1 nuclease protection assay (right) was carried out with the same RNA used in panel B (lane 1) or yeast RNA (lane 2). Arrowhead indicates the TSP of rDNA. (D) Positions of probes (a–e) used in northern blot analysis for 5S rDNA. (E) Mapping of TSP of 5S rDNA by northern blot analysis. Others are the same as in panel B. (F) Sequence of rDNA (RRNb) promoter region. Arrow denotes the TSP.