A cDNA probe was prepared to investigate the regulation of proenkephalin biosynthesis in the rat. This was necessary because human and bovine proenkephalin cDNA were not sensitive enough for the accurate detection of preproenkephalin mRNA in tissues that contain low copy numbers of this message, such as the adrenal gland. The rat probe was prepared in the following manner. Preproenkephalin mRNA was enriched by sucrose gradient centrifugation of poly(A)-containing mRNA from rat brain and was used as a template for double-stranded cDNA synthesis. The resulting cDNA was inserted into the plasmid pBR322, and recombinant plasmids were used to transform Escherichia coli RR1 cells. A synthetic oligodeoxyribonucleotide (30 bases long) with a sequence that had previously been shown to be identical in bovine and human preproenkephalin cDNA was prepared to screen the clone bank. The plasmid with the longest cDNA insert (about 1200 bases) from the positive clones was isolated, and the sequence of the entire protein coding region was determined. Like the bovine and human gene products, rat preproenkephalin contains four [Met]enkephalin sequences and one copy each of [Leujenkephalin, [Met]enkephalin-Arg6-Gly7-Leu8, and [Met]enkephalin-Arg6-Phe7. Rat preproenkephalin is 80% and 83% homologous to the bovine and human forms, respectively, at the nucleotide level and is 82% homologous to both species at the amino acid level. Rat preproenkephalin contains 269 amino acid residues, making it larger than the human (267 residues) and bovine (263 residues) precursors. The sensitivity for detection of rat preproenkephalin mRNA with the rat cDNA was several times greater than with the corresponding cDNAs from bovine and human sources.Since the discovery of the enkephalin pentapeptides nearly a decade ago (1), much has been learned about the biosynthesis of [Met]-and [Leu]enkephalin. In addition to the isolation and sequence determination of many enkephalin-containing peptides from bovine adrenal medulla (see ref. 2 for review), the mRNA from bovine adrenal medulla and human pheochromocytoma, which encodes the precursor protein preproenkephalin, has been cloned and sequenced (3-5).The exact function of the enkephalin-containing peptides is still not known even after many years of intensive investigation. It is likely that knowledge concerning the mechanism of action of these peptides and the regulation of their metabolism will come from research on the rat, the animal that has provided most of our knowledge concerning opiate pharmacology. Studies in our laboratory on the stimulation of proenkephalin synthesis in the rat adrenal gland following denervation (6-8) have been hampered because human and bovine cDNA were found not to be sufficiently sensitive as probes for detecting preproenkephalin mRNA in this rat tissue, which has a low copy number of the message under normal conditions. Rat preproenkephalin cDNA was cloned to provide a homologous and, quite likely, a more sensitive probe for monitoring transcriptional changes in rat ...

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The human leukocyte interferon-alpha L (IFN-alpha L) appears to be a pseudogene because it contains a termination codon in the DNA coding for the precursor peptide (signal peptide), but the remainder of the gene codes for an interferon protein of normal length. To determine if this gene codes for an active interferon molecule, an expression plasmid was constructed for IFN-alpha L that was produced in Escherichia coli. The IFN-alpha L is active on human and bovine cells, and exhibits a trace of activity on mouse cells.