Fig. 2.

Cardiac ganglia express proPACAP mRNA. Total RNA from individual atrial cardiac ganglia preparations was reverse-transcribed, and each of three oligonucleotide primer sets spanning the proPACAP transcript coding region (schematic diagram, top) was used for PCR. The cDNA templates were amplified using primers PCP1 and PCP2, and the products were resolved on 1.6% agarose gels, stained with ethidium bromide, and visualized by UV illumination. A single proPACAP band was produced (left panel, top). The identity of the RT-PCR product was substantiated using sequence-specific hybridization. When the proPACAP amplified products were transferred to Nytran and hybridized to a radiolabeled PACAP oligonucleotide probe internal to the primer sites, a single band of the appropriate size was identified (left panel, bottom). The 445 base pair product amplified using the primer pair PCPX1 and PCPX2 was isolated for diagnostic restriction analyses usingHph I, Ban II, Mse I, andBsfX I (center and right panels). The amplified products were digested with each enzyme for 4 or 16 h, fractionated on agarose gels, stained, and examined under UV illumination. In each instance, digestion with endonuclease generated the predicted cleavage products. Control represents undigested amplified product. The schematic diagram is based on the rat neuronal proPACAP cDNA sequence reported by Ogi and coworkers (1990) (GenBankM63006). Gray, 5′ untranslated region (5′ UTR);white, coding region; black, 3′ untranslated region (3′ UTR); thick lines, regions amplified by the specified primers.