Figure 2.

Mapping of the transcription start site (TSS) of PRL gene in grass carp. (A) Primer scanning to determine the region containing TSS. PCR amplification was performed to scan the possible region using downstream primer PS1 as the anchor primer combined with series upstream primers, respectively. In the PCR reactions, reverse transcribed cDNAs from carp pituitary total RNA were used as the template and parallel PCR with genomic DNA was used as the positive control. PCR with DDW as a template was used as the negative control (−ve). (B) Primer extension to identify the position of transcription start site. Total RNA (20 μg, lane 3) were prepared from steady-state incubated grass carp pituitary cells and hybridized with the [γ-32P] – labeled extension primer PE1. DNA sequencing ladder with PRL promoter (left lanes C, T, A, and G) was also performed to determine the position of reverse transcribed cDNAs. The primer extension samples together with sequencing ladders were size-fractionated with in an 8% polyacrylamide gel to identify the TSS.