Neuropeptides are short peptides that have been shown to act as neuronal signaling molecules in many organisms. The FMRFamide neuropeptide was first discovered as a cardioexcitatory agent in mollusks. Many members of the FMRFamide family have since been identified in organisms throughout the animal kingdom, and have been shown to have varying functions. For instance, physiological studies in Ascaris suum have shown that FMRFamide-related peptides have both inhibitory and excitatory effects on muscle contraction, depending on the particular peptide. At least 17 genes,
flp-1 through
flp-17, encoding different FMRFamide-like peptides have been identified in C. elegans.
flp-2 through
flp-17 were identified by BLAST and GENBANK searches of the C. elegans sequence database. Expression of
flp-1 through
flp-13 was verified using RT-PCR; EST sequences have been isolated for
flp-14 and
flp-15. The focus of my research is to determine the function of a particular flp gene product, FLP-8, in C. elegans.
flp-8 encodes 3 copies of a single neuropeptide, KNEFIRFamide. This peptide has been isolated in other nematodes, including Ascaris.
flp-8 transcripts are detectable by RT-PCR in all developmental stages of C. elegans, except for adult. To examine phenotypes associated with
flp-8 disruption, a
flp-8 deletion mutant was isolated by screening a C. elegans EMS-mutagenized library. The deletion removes the entire
flp-8 presumptive coding region. This deletion strain has been backcrossed into a wild-type background to remove any unlinked EMS mutations. Populations were verified to be homozygous for the
flp-8 deletion by PCR, and are being confirmed by Southern blot analysis. The deletion strain is being assayed for abnormal phenotypes, such as chemosensory, osmosensory, tactile response, locomotory, reproductive, and developmental defects. To localize
flp-8 expression in C. elegans, germline transformation of GFP and lacZ reporter constructs under the control of the
flp-8 promoter are being generated. This localization is further being confirmed through antibody staining of whole worms with a
flp-8 peptide-specific antibody. Finally, the exact 5' and 3'
flp-8 exon junctions are being determined by using primer extension and oligo(dT) PCR, respectively.