No abstract

No abstract

A member of the RPCH/AKH (red-pigment-concentrating hormone/adipokinetic hormone) family of arthropod neuropeptides was identified in the fruitfly Drosophila melanogaster, and its structure was determined by automated Edman degradation and m.s. using fast-atom-bombardment ionization and a tandem hybrid instrument capable of high sensitivity. The sequence of this peptide, which we call ‘DAKH’, is pGlu-Leu-Thr-Phe-Ser-Pro-Asp-Trp-NH2 (where pGlu is pyroglutamic acid and Trp-NH2 is tryptophan carboxyamide). H.p.l.c. analyses of extracts of the three body segments revealed that more than 80% of the peptide is contained in the thorax. Although DAKH is typical of family members in its general structure and distribution in the animal, it is unique in containing a residue which is charged under physiological conditions. The evolutionary significance of this change is considered.

We have used a synthetic deoxydecanucleotide to generate an insulin-specific cDNA probe suitable for selecting transformants that contain nearly full-length cDNAs corresponding to the mRNAs coding for rat insulins I and II. Double-stranded cDNA was synthesized from x-ray-induced rat insulinoma poly(A)RNA, inserted in pBR322 plasmid DNA by the homopolymeric tailing technique, and cloned in Escherichia coli X1776. Colony hybridization with oligonucleotide-primed cDNA yielded 16 positive clones of which 7 corresponded to rat insulin I mRNA and 9 to rat insulin II mRNA. Restriction endonuclease maps of representative clones of each group indicated that these contained the complete coding sequences, as was confirmed by nucleotide sequence analysis of the 5' region of the cloned DNA for rat insulin II. Nucleotide sequence analysis also established the amino acid sequence of the prepeptide of rat preproinsulin II. Comparison of the amino acid sequence of the prepeptides of rat preproinsulin I and II shows that three conservative amino acid substitutions have occurred in this region of the molecule.

We have used a specific deoxyoligonucleotide probe to detect gastrin mRNA in poly(A-enriched RNA preparations from hog antrum. The nucleotide sequence of the oligonucleotide, d(C-T-C-C-T-C-C-A-T-C-C-A), was deduced from the unique amino acid sequence Trp-Met-Glu-Glu of gastrin. antibodies will not precipitate a gastrin-specific translation product produced in vitro.In this communication we describe an approach for detection and characterization of the mRNA for gastrin using an oligonucleotide probe whose sequence is deduced from a unique amino acid sequence of the hormone. The procedure allows the detection and partial sequence analysis of a mRNA species present as about 0.6% by weight of the poly(A)-enriched RNA preparation from hog antral mucosa, or about 0.005% of the total RNA extracted. MATERIALS AND METHODSEnzymes and Reagents. Reverse transcriptase (RNA-dependent DNA polymerase) from avian myeloblastosis virus was provided by J. W. Beard (Life Sciences, Inc., St. Petersburg, FL), and exonuclease-free T4 phage polynucleotide kinase (32,000 units/mg) was isolated by a modified procedure of Panet et al. (10). Oligo(dT)-cellulose was prepared as described by Gilham (11).Preparation of Poly(A)RNA. Antral mucqsa from freshly slaughtered hogs was dissected from the pyloric gland area, wiped free of mucus, and immediately frozen in liquid nitrogen. The frozen tissue (15 g obtained from one stomach) was added to 80 ml of phenol/chloroform/isoamyl alcohol (50:48:2, vol/vol) and mixed with 80 ml of 0.2 M Tris-HCl (pH 9.0)/0.1 M LiCI/25 mM EDTA/1% sodium dodecyl sulfate. The mixture was homogenized with a Polytron (Brinkmann) operated at high speed for several short bursts totaling 1 min, and the phases were separated by centrifugation for 15 min at 15,000 X g. The aqueous phase was removed, and the organic phase and a large interphase were reextracted with an additional 80 ml of buffer. Finally, the aqueous phases were combined and reextracted with 40 ml of phenol/chloroform/isoamyl alcohol. Nucleic acids were precipitated from the aqueous phase at -20°C for 16-24 hr after addition of 0.1 vol of 2 M sodium acetate and 2.5 vol of 95% (vol/vol) ethanol. RNA was further purified by CsCl centrifugation as described by Glisin et al. (12) followed by chromatography on oligo(dT)-cellulose (13).