The pond snail Lymnaea stagnalis is an excellent model system in which to study the neuronal and molecular substrates of associative learning and its consolidation into long-term memory. Until now, the presence of cyclic AMP (cAMP)-responsive element binding protein (CREB), which is believed to be a necessary component in the process of a learned behavior that is consolidated into long-term memory, has only been assumed in Lymnaea neurons. We therefore cloned and analyzed the cDNA sequences of homologues of CREB1 and CREB2 and determined the presence of these mRNAs in identifiable neurons of the central nervous system (CNS) of L. stagnalis. The deduced amino acid sequence of Lymnaea CREB1 is homologous to transcriptional activators, mammalian CREB1 and Aplysia CREB1a, in the C-terminal DNA binding (bZIP) and phosphorylation domains, whereas the deduced amino acid sequence of Lymnaea CREB2 is homologous to transcriptional repressors, human CREB2, mouse activating transcription factor-4, and Aplysia CREB2 in the bZIP domain. In situ hybridization revealed that only a relatively few neurons showed strongly positive signals for Lymnaea CREB1 mRNA, whereas all the neurons in the CNS contained Lymnaea CREB2 mRNA. Using one of the neurons (the cerebral giant cell) containing Lymnaea CREB1 mRNA, we showed that the injection of a CRE oligonucleotide inhibited a cAMP-induced, long-lasting synaptic plasticity. We therefore conclude that CREBs are present in Lymnaea neurons and may function as necessary players in behavioral plasticity.

Insulin-related peptide cDNA was characterized in the Pacific oyster Crassostrea gigas. It was determined that three transcripts with differing lengths of 3 -untranslated region (3 -UTR) were expressed in the visceral ganglia. The insulin-related peptide cDNA contained a number of AUUUA motifs that were typical of adenylate/uridylaterich elements in the 3 -UTR. The deduced preprohormone was a polypeptide of 161 residues and showed a conformation typical of preprohormones of the insulin superfamily, which included conserved amino acids necessary to adopt the globular insulin structure. The expression of the three different transcripts was variable throughout the year, with the highest expression observed in March and lower expression in November and July.

SUMMARY: Based on the results of neuroendocrinological studies on the pond snail Lymnaea stagnalis, insulin-related peptides have been suggested to control normal body growth in gastropod mollusks. To elucidate the presence of a homologous peptide in bivalve mollusks, we clarified the cDNA structure and expression pattern of the insulin-related peptide gene together with the prohormone convertase (PC2) gene of the Pacific oyster, Crassostrea gigas. The deduced amino acid sequence of the peptide showed a preprohormone structure typical of insulin, and conserved the amino acids essential for the tertiary structure. Three different sizes of the peptide mRNA were detected only in the visceral ganglia (VG) where the PC2 gene is also expressed. The predicted Crassostrea PC2 amino acid sequence is 70% identical to Lymnaea PC2 and 59% identical to human PC2. In addition to being expressed in the VG, Crassostrea PC2 is also expressed in the intestine, gill, and mantle.

A cDNA encoding the first mollusk member of the PAR subfamily of bZIP transcription factors has been characterized in the Pacific oyster, Crassostrea gigas. The sequence of the cDNA predicts a protein of 260 amino acids that has 41-50% identity with the PAR domain, 72-79% identity with the basic DNA-binding domain and 34-56% identity with the leucine zipper domain of other members of the PAR subfamily. Polymerase chain reaction with reverse transcription indicates that this gene is expressed during developmental stages from an unfertilized egg to a juvenile. It was demonstrated by northern hybridization that the gene is also expressed on several adult tissues. The identification of a novel member of the PAR subfamily bZIP genes in mollusks may help to identify common functions that have been conserved through evolution and to elucidate evolutionary relationships within this subfamily of proteins.