Cardioactive neuropeptide Phe-Met-Arg-Phe-NH2 (FMRFamide) and novel related peptides are encoded in multiple copies by a single gene in the snail Lymnaea stagnalis

Abstract

The neuropeptide Phe-Met-Arg-Phe-NH2 (FMRFamide) is a potent cardioactive neuropeptide in Lymnaea stagnalis. Isolation and sequencing of 2 cDNAs and a genomic clone shows that a single gene encodes a precursor protein which contains 9 copies of the FMRFamide peptide, 2 copies of the related peptide Phe-Leu-Arg-Phe-NH2 (FLRFamide), and single copies of the putative pentapeptides Gln-Phe- Tyr-Arg-lle-NH2 (posttranslationally modified to pQFYRlamide) and Glu- Phe-Leu-Arg-lle-NH2 (EFLRlamide). The gene is transcribed in the CNS and gives rise to a single RNA of 1.7 kb in size. The organization of the Lymnaea gene is significant with respect to the evolution of FMRFamide and related peptides in other organisms.

Myomodulin Gene ofLymnaea: Structure, Expression, and Analysis of Neuropeptides

“…The most likely cleavage site, RFRR (residues 114 -117), is found within the spacer sequence between the first two myomodulins, GLQMLRLamide and QIPMLRLamide. This putative cleavage site is similar to the tetrabasic cleavage sequence observed in both the Lymnaea FMRFamide (Linacre et al, 1990) and the Aplysia ELH precursor proteins, where it has been demonstrated to be the first site of processing, allowing the differential sorting and trafficking of peptides encoded at opposite ends of the same precursor protein (Sossin et al, 1990). A possible cleavage site is also present at an equivalent position in the Aplysia myomodulin precursor (Lopez et al, 1993;Miller et al, 1993).…”

“…Cleavage at this point would produce a 330-amino-acid propeptide that encodes five different myomodulin-like peptide sequences. These are flanked at the N terminus by lysine-arginine basic amino acid pairs (sites of endoproteolytic cleavage), with the exception of the first predicted peptide sequence, GLQMLRLG, which is preceded only by a single arginine residue; endoproteolytic cleavage at such sites is also well documented (Newcomb et al, 1987;Linacre et al, 1990). All of the putative peptides are flanked at the C terminus by glycine residues followed by a pair of basic amino acids.…”

Genomic organization of the FMRFamide gene in Lymnaea: multiple exons encoding novel neuropeptides

“…It had previously been shown that the tetrapeptide FMRFamide (Linacre et al, 1990) and the heptapeptides GDPSDPFLRFamide (Saunders et al,199 1) were encoded by separate exons and that both exons are spliced onto a common 5' exon that encodes a hydrophobic leader sequence (Saunders et al, 1992). The heptapeptide cDNA sequence was not complete as a PCR product containing only the 5' end of the cDNA had been isolated and sequenced.…”

“…Analysis of genomic clones By analyzing a XEMBL 3 recombinant clone it had already been established that the tetra-and heptapeptides are encoded by exons located on separate EcoRI fragments of 4.1 kb and 3.4 kb, respectively (Linacre et al, 1990;Saunders et al, 199 1). Detailed restriction and sequence analysis has shown that the tetrapeptide exon is 1170 bp in length and is separated from the heptapeptide exon of 672 bp, by an intron of 2.9 kb (GenBank accession no.…”

“…Central neuronal actions by this peptide are also well known in mollusks (Abrams et al, 1984;Cottrell and Davis, 1987). The cloning of DNA sequences that encode FMRFamide and related peptides from a number of invertebrate organisms has shown that these peptides are often highly repeated within a protein precursor and are found together with a number ofnovel peptides (Aplysia, Schaefer et al, 1985;Drosophila, Nambu et al, 1988;Schneider and Taghert, 1988; Lymnaea stagnalis, Linacre et al, 1990;Saunders et al, 1991;Calfiactis, Darmer et al, 1991;Helix, Lutz et al, 1992; Caenorhabditis elegans, Rosoff et al, 1992).…”