FIG 4.

Mapping of the 5′ end of the ORF52 transcript by S1 nuclease protection assay. (A) S1 nuclease protection assay. Total RNA from uninfected (lane 3) and infected (lanes 1 and 2) BHK-21 cells was hybridized with a labeled probe and digested with 200 U (lane 1) or 400 U (lanes 2 and 3) of S1 nuclease, and the products were separated on a denaturing gel along with markers of 29 nt (lane 4) and 31 nt (lane 5) as well as the undigested probe (lane 6). (B) Nucleotide sequence of the probe and surrounding region. The translational start codon of ORF52 is indicated by underlined bold uppercase letters. A gray box represents the 15-bp ORF52 promoter region defined by the reporter studies (Fig. 2), with the TATA box highlighted by underlined italic bold letters. The probe used in the S1 nuclease protection assay is indicated by a dashed line, and the two triangles show the two mapped transcription sites.