FIG. 4.

Transcription start site of the α-MPP gene. (A) Primer extension mapping of the transcription start site. An 18-nt primer, complementary to nt +146 to +163, was 5′ end labeled with [γ-³²P]ATP and hybridized to 50 μg of total RNA from B. emersonii vegetative cells (lane 1), zoospores (lane 2), and germling cells (lane 3). The hybrids were then extended with reverse transcriptase, and the extension products were resolved by denaturing gel electrophoresis and autoradiography. The sequencing ladder was generated with the same 18-nt oligonucleotide as a primer and M13mp19 containing the 5′ end of the α-MPP gene (coding strand). (B) Nucleotide sequence of the 5′ region of the α-MPP gene. Nucleotide +1 denotes the A of the ATG encoding the initiator methionine. The underlined region is complementary to the oligonucleotide used for the primer extension experiment. The predicted transcription start point is indicated by an arrow. The putative core sequences representing the binding sites for the TATA-binding protein (TATA box), Sp1 (GC box), CTF/NF1 (CCAAT box), and helix-loop-helix transcription factor (E box) are indicated.