Determination of the transcriptional start site of the mvWF gene. (A) In primer extension assays, purified total RNA from mouse lung tissue (lanes 1 and 2) was used as template. Primer extension analysis was performed using a 32P-labeled antisense oligonucleotide spanning the region between +114 and +153 and AMV reverse transcriptase. DNA size markers and a dideoxy sequencing reaction of mvWF-pUC19-5 generated with the same primer are included on the left. The arrow indicates the start site of transcription. (B) In RT-PCR analyses, first-strand cDNA synthesis and PCR reactions were performed with total RNA from mouse lung (lanes 2, 6, 9, and 12) and heart (lanes 3, 7, 10, and 14) tissue. P1-7910 DNA template was included as a positive control (lanes 1, 5, 8, and 11). A 100-bp DNA ladder is shown in lanes 4 and 11. The location of the primers relative to the transcriptional start site (depicted by an asterisk) is shown above. The primer sequences are described in Materials and Methods. PCR-generated fragments from cDNA were obtained only with primer sets D-E (144 bp) and C-E (232 bp).
