Characterization of the gene for leucomyosuppressin and its expression in the brain of the cockroach Diploptera punctata

Using HPLC separation, radioimmunoassay, and subsequent bioassay, we have detected the presence of an active peptide, which co-elutes with the insect myoinhibitory peptide leucomyosuppressin, in the brain of the cockroach Diploptera punctata, We have isolated a cDNA encoding the precursor for this peptide from cDNA libraries representing D. punctata brain RNA, The cDNA sequence contains an open reading frame that upon translation would result in a prepropolypeptide of 96 amino acids. Proteolytic cleavage of the predicted precursor could result in several peptides, including a 10 amino acid C-terminal peptide that would, upon modification of the NH2 and COOH-terminal amino acids, be identical to the insect FLRFamide, leucomyosuppressin, No other RFamide products are predicted to be processed from the precursor, Southern blot analysis indicates that the gene is present in the D. punctata genome in a single copy, Northern blot analysis shows that the gene is predominantly expressed as a 3.8 kb mRNA in cockroach brain. Study of the expression of the leucomyosuppressin gene in D. punctata brain, using in situ hybridization, indicates that expression occurs primarily in the pars intercerebralis of the protocerebrum, a region showing abundant FMRFamide-like immunoreactive neurosecretory cells, Immunohistochemistry and HPLC coupled to radioimmunoassay indicates that leucomyosuppressin represents a significant proportion of FMRFamide-related peptide production in the brain, However, HPLC analysis also indicates the presence of significant levels of other related peptides, demonstrating the presence of more than one FMRFamide-related gene in this insect. Crown copyright (C) 1996 Published by Elsevier Science Ltd.