Fig. 4
From: Molecular Cloning of the Human Leukotriene C4 Synthase Gene and Assignment to Chromosome 5q35
Determination of transcription initiation site
Total RNA was isolated from THP-1 cells and was annealed with a 25-bp reverse primer corresponding to nucleotides 76 to 100 of the genomic sequence and labeled with T4 polynucleotide kinase with [γ-32P] ATP. The samples were then reverse transcribed using Superscript II. Electrophoresis of these samples was performed in 8% denaturing Polyacrylamide and the gel was subjected to autoradiography. The left lane (−) lacks RNA (negative control). The right lane (+) is the same reaction conducted with 44 µg of total RNA. The primer extension product was found to be 100 bp. This size was confirmed by repeat gels run in parallel with a sequencing reaction using the P1 plasmid and the same primer. By comparison to labeled markers, the transcription initiation site mapped to a single site 78 bp upstream of the ATG initiator codon.