Isolation and characterization of the xenopus laevis cdna and genomic homologs of neuropeptide Y

Abstract

We have isolated Xenopus laevis cDNA and genomic clones encoding the neuropeptide Y (NPY) mRNA and gene using a probe from the human NPY gene. The longest open reading frame in the cDNA encodes a peptide 76% identical to human prepro-NPY and 73% identical to rat prepro-NPY. The putative mature Xenopus NPY (XNPY) peptide is 94% identical to both human and rat peptides. A genomic clone containing 422 base pairs of 5'-flanking sequences and the 5'-end of the mRNA was also isolated. Primer extension analysis was used to map the transcription initiation site of the Xenopus NPY gene. Comparison of the 5 '-flanking sequences of the Xenopus laevis, human, and rat NPY genes resulted in areas of high conservation, including the TATA box and the CT box previously shown to interact with Spl-like proteins. Distribution of the Xenopus NPY message was analyzed by Northern analysis and RNAse protection. XNPY transcripts were not detected in whole developing embryo RNA, but were detected in adult frog brain RNA. We have also conducted preliminary studies of the XNPY promoter, utilizing an XNPY/chloramphenicol acetyl-transferase fusion construct. This study has demonstrated that Xenopus NPY shares a high degree of identity to its human and rat counterparts and that this homology extends to the gene, which contains similar cis-elements positioned near the transcription start site.