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A synthetic DNA based on the known amino acid sequence of the brain/gut peptide cholecystokinin (CCK) was synthesized. This DNA contained deoxyinosines at ambiguous codon positions and was used as a probe to isolate the CCK gene directly from a human genomic library. Nucleotide sequence analysis of the isolated gene revealed that human preprocholecystokinin consists of 115 amino acid residues, with 11 amino acids in common with the human gastrin precursor, another member of the gastrin-CCK family, and that the coding region is separated by a single, long intron. CCK appears to be encoded by a single-copy gene in the haploid human genome, as revealed by genomic Southern hybridization analysis, suggesting that the same gene is expressed both in gut and brain.Cholecystokinin ( Recently, the amino acid sequence of porcine CCK58 and the cDNA sequence for rat CCK have been reported (4, 5).Considering the large accumulation of data on human CCK (its suspected biological role in the brain, its possible connection with diseases, etc.), it is very important to determine the amino acid sequence of human CCK and its precursor. The carboxyl-terminal five amino acid residues of CCK are identical with those of gastrin (6), another brain/gut peptide, which induces the release of gastric juices and which is detected also in the brain, although its function there is not known. These two peptide hormones, in addition to caerulein found in frog skin (7), comprise the gastrin-CCK family. The related structures of these peptide hormones are also of interest in terms of molecular evolution. These concerns prompted us to isolate and study the human CCK gene.Lack of a CCK-producing tumor cell line precluded our obtaining human CCK mRNA for analysis, because its concentrations in the available human tissues are extremely low. We therefore isolated the gene directly from a genomic library by using an appropriate oligonucleotide probe. For cDNA cloning (8), mixed-sequence oligonucleotide probes of 14-17 nucleotides often have been used. These probes typically consist of mixtures of 8-32 oligodeoxynucleotides, in order to represent every possible codon combination for the stretch of amino acid sequence in question. For the direct cloning of a single-copy gene in such a complex population as a human genomic library, however, a much longer probe is needed (9). One difficulty is that mixed probes cannot be used effectively because they necessarily contain too many varieties of molecular species due to many codon degeneracies. We overcame this problem by using an appropriate base analog that can "pair" with any of the four natural bases at the ambiguous positions, with or without forming hydrogen bonds. Probes with inosines at the ambiguous positions in the degenerate codons were useful for this purpose. Using this novel probe, we isolated the human CCK gene directly from the genomic library and determined its primary structure.MATERIALS AND METHODS Oligonucleotide Probe. The deoxyinosine-containing oligodeoxynucleotide probe was syn...

BaciUus brevis 47 contains two cell wall proteins termed the outer wall protein (OWP) and middle wall protein (MWP), each of which forms hexagonal arrays in the cell wall. A 6-kilobase Bglll-Bcll fragment of B. brevis 47 DNA cloned into BaciUus subtilis with a derivative of pHWl as a vector directed the synthesis of a polypeptide, with almost the same molecular weight as the authentic OWP, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which was specifically recognized by the anti-OWP antibody. Nucleotide sequence analysis of the subfragment revealed that it contains two open reading frames in tandem.The upstream truncated open reading frame corresponds to the carboxy-terminal portion of the MWP, and the downstream open reading frame corresponds to the entire translational portion of the OWP. The latter encodes a secretory precursor of the OWP, consisting of a total of 1,004 amino acid residues with a signal peptide of 24 amino acid residues at its amino-terminal end. Furthermore, analysis of transcripts in B. brevis 47 suggests that the MWP and OWP genes, in that order, constitute a cotranscriptional unit and that the major promoter shared by the two genes is located upstream of the MWP gene.

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