Characterization of Xenopus laevis proenkephalin gene

Abstract

Enkephalins are opiate peptides found in a variety of tissues including brain and pituitary. In brain, they function as neurotransmitters, neuromodulators and neurohormones. Recent studies show that proenkephalin mRNA is expressed early in development both in mammals and the amphibian, suggesting that enkephalins may play a unique role in embryogenesis. In order to characterize factors which regulate the onset and patterning of expression of this gene in adult and developing frog embryos, the proenkephalin A gene was cloned from Xenopus laevis. The clones have been characterized by DNA sequencing and restriction endonuclease mapping. The gene is made up of three exons which span approximately 12 kb. Exon I encodes the 5′ untranslated region of the mRNA. Exon II contains the signal peptide and the N terminus of the mature protein. Biologically active opioid peptides are generated from exon. III. Comparison to mammalian proenkephalin genomic sequence indicated that nucleotide sequences of the 5′ flanking region, noncoding exon I and exon II were not well conserved but exon III was highly conserved. Primer extension and RNase protection assay analyses of the RNA transcripts revealed two major 5′ ends. The putative TATA box, CAAT box, CRE and Pit 1 elements have been identified on this gene by sequence homology to published consensus sequences. To assay for sequences that could potentially regulate Xenopus proenkephalin expression, we transfected constructs that contained upstream genomic sequences linked to the CAT reported gene into various eukaryotic cell lines. The expression of the fusion gene constructs were detected and could be induced 10- to 30-fold upon treatment with forskolin. Although the Xenopus proenkephalin gene promoter does not share sequence homology with the mammalian promoter, it was found to be transcriptionally active upon transfection into mammalian cell lines, indicating that at least some of the transacting factors required for expression are conserved through evolution.