A cDNA clone encoding neuropeptides isolated from Aplysia neuron L11.

Abstract

Single nerve cells can use more than one substance as extracellular chemical messengers. Classical transmitters have been shown to coexist in the same neuron and possibly even in the same vesicle as neuroactive peptides. Furthermore, multiple neuroactive peptides, which are thought to be coreleased, are often encoded in the same precursor assuring stoichiometric synthesis. The precise organization of multiple message systems and the physiological significance of the coexistence is poorly understood. The abdominal ganglion of the gastropod mollusc Aplysia contains a number of identified neurons that are cotransmitter candidates. One such cell, Lii, is cholinergic and probably also uses biologically active peptides. Differential screening with labeled cDNA was used to isolate cDNA clones expressed specifically in the bag cells and abdominal ganglion neurons L11 or R15. Analysis of an L11-specific clone suggests that it encodes a 14.7-kDa protein that is the precursor for the secreted peptides. The poly(A)+ RNA transcript is -1.2 kilobases and there are 1-3 copies of this gene in the Aplysia haploid genome.The behavioral potential of an organism is determined by the specific interactions between cells of the nervous system. Intercellular communication in the brain is generated by two general mechanisms. First, a presynaptic neuron can send an axon to an appropriate position and release a chemical messenger, which traverses a short distance to bind a receptor on a target cell. The specialized membrane structures at the synapse juxtapose release sites with receptors and often include efficient mechanisms for removing the transmitter from the synaptic cleft. The chemical messengers at synapses are normally classic neurotransmitters, such as acetylcholine and y-aminobutyric acid. Neurons can also influence electrical and metabolic activities of target cells some distance from the site of messenger release. This form of intercellular communication is often mediated by biologically active peptides, which are carried to target tissues through the circulation or simply through passive diffusion. The specificity of these interactions is not determined by spatial constraints but by the distribution of highly specific receptors for the messengers. In addition, the concentration of this type of extracellular messenger necessary to achieve a biological response is often several orders of magnitude lower than that of classical transmitters. Thus, in thinking of neural circuitry, one must superimpose these two forms of communication between cells to generate an accurate representation of neuronal interactions. These two general forms of intercellular communication are of course not as distinct as outlined above. Recent investigations suggest that a single neuron may use more than one transmitter (1), frequently a classic neurotransmitter and a peptide, to elicit a variety of responses (2, 3).To clarify these and other issues, we use the gastropod Fig. 1). In this report, we extend these studies to a number of other pepti...

The small cardioactive peptides A and B of Aplysia are derived from a common precursor molecule.

“…The coding region is flanked by 111 nucleotides on the 5' end and by an 871-base-pair 3' untranslated region. Both the 3' untranslated region in this clone and in other characterized Aplysia transcripts contain long purine-and pyrimidine-rich stretches (12,13). There are also three A-N-U-A-A-A (where N is any nucleotide) sequences in this region.…”

“…Aplysia neuropeptides are particularly amenable to this approach since peptidergic cells can be identified and individually dissected. In these cells the peptide precursor and the mRNA encoding it are a significant proportion of the total protein and mRNA, thus facilitating molecular genetic and biochemical studies (11)(12)(13)(14)(15). Biochemical-bioassay studies (4) and the profile of SCPB immunoreactivity presented above suggests that the buccal ganglia, and especially the neurons B1 and B2, are a rich source of SCPB-precursor mRNA.…”

Release of pedal peptide from Aplysia neurons in primary culture

“…These results suggest that this may be a useful procedure for determining the nature of peptides released from other peptidergic neurons in Aplysia. A promising application for this technique might include the very large neurons in the abdominal ganglia of Aplysia for which the sequences of the mRNA that encode peptide precursors have been determined, but the nature of the mature released neuropeptide has not (Taussig, Kaldany, and Scheller, 1984;Shyamala, Fisher, and Scheller, 1986).…”

Aplysia californica neurons express microinjected neuropeptide genes.

“…The pRL-2A plasmid was constructed in two steps. First, a 192-base-pair (bp) HaeIII partial digest fragment (nucleotides 376 to 568 [48]) isolated from Lli cDNA was isolated and ligated at the AluI site (nucleotide 277 [39]) of the subclone pMP65 (49), which contains the second exon of the R14 gene. Second, one clone which contained the Lii insert in the proper orientation was purified, and the EcoRlSstI fragment was substituted for the corresponding fragment in the R14 genomic clone.…”

“…RESULTS Aplysia neuropeptide gene promoters. Genomic and cDNA clones encoding neuropeptide genes expressed in R3-14, Lii, and the bag cell neurons had previously been isolated and characterized (39,45,47,48). The transcribed regions spanned a minimum of 7 kilobases (kb) in the case of the R14 peptide gene and over 30 kb in the case of the Lii peptide gene.…”