FIGURE 3.

5′-end mapping of RNAs transcribed from the fission yeast phosphate regulon. (A) Schematic illustration of the adjacent pho84⁺ (SPBC8E4.01c) and pho1⁺ (SPBP4G3.02) genes on fission yeast chromosome II, with the open reading frames denoted by arrows in the direction of mRNA synthesis. The experimentally mapped 5′ ends of the pho84 and pho1 mRNAs and the intervening noncoding prt RNA are indicated by their distance (nt) from the translations start sites of the pho84 and pho1 mRNAs. (B) To map the 5′ ends of the pho84 and prt RNAs, we analyzed the reverse transcriptase primer extension product (lanes *) in parallel with a series of DNA-directed primer extension reactions that contained mixtures of standard and chain-terminating nucleotides (the chain terminator is specified above the lanes). The primer extension products were analyzed by electrophoresis through a 42-cm denaturing 8% polyacrylamide gel and visualized by autoradiography of the dried gel. The 5′ RNA ends are denoted by ◂ to the right of the gel. The coding strand DNA sequences flanking the pho84 and prt transcription start sites are shown at right, with the start sites denoted by ↱. (C) ³²P-labeled oligonucleotide primers complementary to pho1 and act1 mRNAs (left panel), pho84 mRNA RNA (top right panel), or prt RNA (bottom right panel) were annealed to total RNA from pho7⁺ (WT) or pho7Δ strains and extended with reverse transcriptase. The reaction products were analyzed by denaturing PAGE and visualized by autoradiography.