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Based on the sequencing of genomic and cDNA clones, we were able to determine that the FMRFamide gene consists of five exons covering at least 20 kb and predict the presence of further novel peptides. The exons are alternatively spliced: exon I (hydrophobic leader sequence) to exon II (tetrapeptides) and exon I to exons III (heptapeptides), IV, and V. A cDNA clone encoding the heptapeptides is described and has also been shown to encode further novel peptides SKPYMRFamide, HDYMRFamide, and SSFPRYamide. Analysis of the right internal parietal nerve using mass spectrometry showed that the novel peptide SKPYMRFamide was cleaved from the precursor. This peptide excites neurons, suggesting a physiological function in the CNS.

The FMRFamide gene of the snail Lymnaea encodes tetrapeptides (FMRFamide/FLRFamide) and heptapeptides (GDPFLRFamide/SDPFLRFamide) on separate exons. In situ hybridization probes specific to these exons were used to map the expression of the two exons in identified neuronal systems of the CNS. Analysis of more than 200 preparations showed that cytoplasmic expression of mRNA was exclusively of one type, with individual neurons expressing either the tetrapeptide or heptapeptide exon. Of the approximately 340 neurons expressing the two exons, the majority (80%) expressed the tetrapeptide exon. The tetrapeptide exon was more widespread, occurring in neurons from all 11 ganglia of the CNS. The heptapeptide was mainly confined to two ganglia (visceral and right parietal), with a small number of cells in three other ganglia. Mapping studies combined with dye marking of identified neurons showed the presence of the tetrapeptide exon in several behaviorally important networks: heart motoneurons, whole body withdrawal response motoneurons, and probably penis motoneurons as well as giant identified neurons (LP1, RPD1). The heptapeptides were prominent in two main clusters of cells (Bgp and Fgp) together with a smaller number of tetrapeptide-expressing cells.

Biochemical analysis has shown the pond snail Lymnaea stagnalis to contain 2 main classes of Phe-Met-Arg-Phe-NH2 (FMRFamide)-like neuropeptides: the tetrapeptides FMRFamide and Phe-Leu-Arg-Phe-NH2 (FLRFamide), and the heptapeptides Gly-Asp-Pro-Phe-Leu-Arg-Phe-NH2 (GDP-FLRFamide) and Ser-Asp-Pro-Phe-Leu-Arg-Phe-NH2 (SDPFFRFamide). By genomic mapping and DNA sequencing, we show here that the GDP/SDPFLRFamide coding region lies 3' to the FMRFamide coding region. The absence of an initiating start methionine and the presence of good-concensus 3' and 5' splice sites suggests that the GDP/SDPFLRFamide coding region makes up 1 exon of a larger gene. In addition to 7 copies of GDPFLRFamide and 6 copies of SDPFLRFamide, the exon encoding the heptapeptides also encodes 3 novel peptides, Glu-Phe-Phe-Pro-Leu-NH2 (EFFPLamide), Ser-Asp-Pro-Tyr-Leu-Phe-Arg-NH2 (SDPYLFRamide), and Ser-Asp-Pro-Phe-Phe-Arg-Phe-NH2 (SDPFFRFamide). In contrast to the tetrapeptide FMRFamide precursor protein, the GDP/SDPFLRFamide peptides are encoded contiguously, being separated only by single basic amino acids.