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 synthesis of a complementary DNA copy (cDNA) of hen ovalbumin mRNA using AMV RNA-directed DNA polymerase was studied under different conditions of salt, deoxyribonucleotide concentrations, temperature, and time. It was observed that in the absence of monovalent cation at 46 degrees C a complete transcript of ovalbumin mRNA could be effected by the enzyme. The minimum deoxyribonucleotide requirement for complete synthesis was 35 muM for dATP, dGTP, and dCTP and 200 muM dTTP. By a number of different experimental criteria which included sedimentation on alkaline sucrose gradients and electrophoresis in polyacrylamide gels containing 98% formamide, direct electron microscope visualization, and protection of ovalbumin [25I]mRNA from nuclease digestion it could be demonstrated that a considerable fraction of a complete mRNA transcript was indeed synthesized. The cDNA/ovalbumin mRNA hybrid had a Tm on hydroxylapatite of 92 degrees C, indicating the synthesis of a RNA transcript with a high fidelity. When such a complete ovalbumin [3H]cDNA was synthesized with a specific activity of 10(8) cpm/mug and hyfridized to an excess of chick DNA, the kinetics of hybridization indicated that the cDNA was comprised of a nonrepetitive sequence.

When pBR322 plasmid-harboring Escherichia coli strains RR1 or X1776 were grown in the presence of 1 mg of uridine or cytidine per ml and later treated with chloramphenicol, as much as three times more plasmid deoxyribonucleic acid was recovered than would normally be obtained by routine plasmid amplification procedures.

Molecular cloning has established the primary structures of two precursors of the human pancreas growth hormone-releasing factor (hpGRF-44), somatocrinin. Both polypeptides contain the sequence of hpGRF-44 flanked by basic processing sites. Furthermore, the precursors include a putative signal sequence and a carboxyl-terminal amidation signal for hpGRF-44. The two forms of mRNA code for pre-pro-GRF-107 and pre-pro-GRF-108. Pre-pro-GRF-108 differs from pre-pro-GRF-107 by the insertion of a serine in the carboxyl-terminal portion of the precursor. In vitro translation of tumor poly(A)+ RNA followed by immunoprecipitation with GRF-specific antiserum and gel electrophoresis showed the molecular weight of preprosomatocrinin to be approximately 13,000, which is in good agreement with the molecular weight deduced from the sequences of the cDNA clones.A 44-amino acid peptide (hpGRF-44, somatocrinint) with high intrinsic growth hormone-releasing activity in vitro and in vivo was recently isolated from a human pancreatic tumor and characterized (1, 2). Preliminary biological (3), immunological (4), and physico-chemical (unpublished data) evidence indicates that the tumor-derived hpGRF-44 is identical to hypothalamic growth hormone-releasing factor. The availability of tumor material, highly enriched in the growth hormone-releasing factor compared with hypothalamic tissue, has permitted the molecular cloning and structural characterization of the mRNA encoding hpGRF-44. MATERIALS AND METHODSPoly(A)+ RNA was obtained from 15 g of tumor tissue (stored in liquid nitrogen or at -80°C) by using the guanidine thiocyanate procedure (5) and chromatography on oligo(dT)-cellulose (6). Double-stranded cDNA was synthesized by using a modification of a published method (ref. 7; unpublished data). A cDNA library was constructed by homopolymer tailing of the cDNA with dGTP, hybridizing it to EcoRV-cut pBR322 that had been tailed with dCTP and transforming Escherichia coli RR1 with the chimeric plasmid (8). The solid-phase phosphotriester method (9) was used for the synthesis of two mixed oligodeoxynucleotide probes (see Fig. 1), which were labeled at their 5' ends with [y-32P]ATP (10). The tetradecamer probe was used for colony screening (11) as follows: hybridizations were carried out in 0.75 M NaCl/0.075 M sodium citrate/0. 1% NaDodSO4/ 0.2% bovine serum albumin/0.2% polyvinylpyrrolidone/0.2% Ficoll/partially hydrolyzed yeast RNA (100 ug/ml) at 300C for 2 hr using 1 pmol of labeled probe per ml. The filters were quickly rinsed in 0.3 M NaCl/0.03 M sodium citrate at room temperature. After a high-stringency wash in 0.6 M NaCl/0.06 M sodium citrate for 60 min at 40'C, the filters were exposed to x-ray film overnight.DNA sequence analysis, Southern and RNA blot analyses, nick-translation, primer extension, and NaDodSO4/polyacrylamide and denaturing agarose gel electrophoresis were carried out according to standard procedures (10,(12)(13)(14)(15)(16)(17)(18). Antibodies to hpGRF were produced in rabbits following immunization with...