Gastric inhibitory polypeptide (GIP) is a 42-amino acid hormone that stimulates insulin secretion in the presence of glucose. Complementary DNA clones encoding human GIP were isolated from a library prepared with RNA from duodenum. The predicted amino acid sequence indicates that GIP is derived by proteolytic processing of a 153-residue precursor, preproGIP. The GIP moiety is flanked by polypeptide segments of 51 and 60 amino acids at its NH2 and COOH termini, respectively. The former includes a signal peptide of about 21 residues and an NH2-terminal propeptide of 30 amino acids. GIP is released from the precursor by processing at single arginine residues. There is a region of nine amino acids in the COOH-terminal propeptide of the GIP precursor that has partial homology with a portion of chromogranin A as well as pancreastatin.Gastric inhibitory polypeptide (GIP) was first isolated from porcine small intestine (1, 2) on the basis of its ability to inhibit histamine-stimulated gastric acid secretion in the dog stomach (3). The sequences of porcine (4, 5), bovine (6), and human GIP (7) have been determined; each has 42 amino acids, and the sequence is highly conserved. The porcine and bovine peptides differ from the human at two and four sites, respectively. The sequence of GIP indicates that this peptide is a member of a family of structurally related hormones that includes secretin, glucagon, vasoactive intestinal peptide, and growth hormone-releasing factor (8-10). Subsequent studies of the physiological properties of GIP revealed that it was a relatively poor inhibitor of gastric acid secretion (11) but was a potent stimulator of insulin secretion (12, 13). It is able to stimulate insulin secretion at physiological concentration only in the presence of glucose (14,15). Thus, GIP by functioning as a glucose-dependent insulin-releasing peptide could have a role in maintaining glucose homeostasis. Moreover, it might also be of use in treating some diabetics. As a first step in studying the biosynthesis of this potentially important regulator of beta cell function, we have isolated and sequenced cDNA clones encoding the human GIP precursor. This report describes the sequence of these clones and the predicted amino acid sequence of the human protein. Fig. 1 were synthesized by a modification of the triester method (21). Probes I and II were end-labeled at the 5'-terminal end with T4 polynucleotide kinase (Toyobo, Osaka, Japan) and [y-32P]ATP (Amersham; 6000 Ci/mmol; 1 Ci = 37 GBq) and was purified by passing through a Sephadex G-50 (Pharmacia) column. MATERIALS AND METHODSColony Screening. Escherichia coli HB101 was transformed with the recombinant DNA by the calcium-shock procedure (22). Colony hybridization (23) was performed with probe I (14-mer) at 36°C for 18 hr and with probe II (17-mer) at 40°C for 18 hr in 4x NaCl/Cit (lx = 0.15 M NaCI/0.015 M sodium citrate, pH 7) containing 1OX Denhardt's solution (1x = 0.02% polyvinylpyrrolidone/ 0.02% Ficoll/0.2% bovine serum albumin), 0.1% NaDodSO4, 25 ,ug of...

The sppA gene codes for protease IV, a signal peptide peptidase of Escherichia coli. Using the gene cloned on a plasmid, we constructed an E. coli strain carrying the ampicillin resistance gene near the chromosomal sppA gene and an sppA deletion strain in which the deleted portion was replaced by the kanamycin resistance gene. Using these strains, we mapped the sppA gene at 38.5 min on the chromosome, the gene order being katE-xthA-sppA-pncA. Although digestion of the signal peptide that accumulated in the cell envelope fraction was considerably slower in the deletion mutant than in the sppA+ strain, it was still significant, suggesting the participation of another envelope protease(s) in signal peptide digestion.

Rat liver contains two isozymes of fumarase, mitochondrial and cytosolic enzymes. Recently, we suggested that the precursors of both isozymes might be synthesized by one species of mRNA [Suzuki, T., Sato, M., J . Biol. Chem. 264, 2581-25861. To examine this possibility, we have isolated and characterized rat genomic clones for fumarase. The isolated clones covered almost all of the 5' half of the fumarase gene consisting of five exons. The first exon contained the whole 5' non-coding region and the signal peptide of mitochondrial precursor. The second exon encoded 45 amino acid residues of both mature proteins, starting from the N-terminal alanine. By using the boundary region of the first intron and the second exon as an S1-nuclease-analysis probe, we obtained conclusive evidence that rat liver contains no other mRNA specific for the cytosolic isozyme of fumarase. Two transcription-initiation sites were identified by further S1-nuclease-mapping analysis and were shown to be located very close to each other, differing by only four bases in length. Therefore, these sites were considered to be functionally the same. The results obtained by hybridselected translation, with a DNA fragment of the 5' non-coding region as a hybridization probe for selecting mRNA, were consistent with the above findings. We found a plausible secondary structure within the 5' non-coding mRNA sequence that may impede initiation and so alter the efficiency of translation. We also discuss the mechanism regulating translational initiation.We have reported that rat liver contains two isozymes of fumarase, present in almost equal amounts in the mitochondrial and cytosolic fractions [ l~ 41. The mitochondrial and cytosolic isozymes are synthesized with and without a presequence peptide at the N-terminus, respectively [5], but the primary structure of the two mature enzymes is the same [6, 71, except that the N-terminal amino acid of the cytosolic isozyme is acetylated [4].Recently, we isolated a cDNA clone (pRFum9) for rat liver fumarase containing the whole coding region of the precursors of both the mitochondrial and cytosolic isozymes, but only 25 nucleotides of the 5' non-coding region [6]. This clone contains two in-phase AUG codons separated by a nucleotide segment encoding the signal peptide of the mitochondrial precursor. This characteristic structure of fumarase mRNA together with experimental results obtained by hybridselected translation and primer-extension studies suggested that the two isozymes are translated from a single mRNA by alternative use of the two in-phase translation-initiation sites

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