Molecular cloning of human gastrin cDNA: evidence for evolution of gastrin by gene duplication.

Boel, Esper; Vuust, Jens; Norris, Fanny; Norris, Kjeld; Wind, Anette; Rehfeld, Jens F.; Marcker, Kjeld A.

doi:10.1073/pnas.80.10.2866

Cited by 174 publications

(83 citation statements)

References 31 publications

(21 reference statements)

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“…Double-stranded Aspergillus niger cDNA was synthesized and cloned on pBR327 in E. coli by procedures as described (2). Details of the cloning and the identification of glucoamylase G1 specific recombinants in the resulting cDNA library will be presented elsewhere,…”

Section: Construction and Cloning Of Doublestranded Glucoamylase G1 Cdnamentioning

confidence: 99%

The complete amino acid sequence of the glycoprotein, glucoamylase G1, from Aspergillus niger

Svensson

Larsen

Svendsen

et al. 1983

Carlsberg Res. Commun.

189

158

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The primary structure ofglucoamylase G1 (EC 3.2.1.3) from Aspergillus niger has been determined. Fragments of GI were obtained by cleavage with either cyanogen bromide, hydroxylamine, or S. aureus V8 protease. The resulting peptides were separated using ion exchange chromatography on DEAE-Sephacel, gel filtration, and affinity chromatography on Con A-Sepharose. Secondary fragments were generated by cleavage with either o-iodosobenzoic acid or BNPS-skatole as well as by digestion with S. aureus V8 protease, trypsin, and endoproteinase Lys-C. These peptides were purified by the procedures mentioned above and by reverse phase HPLC. The present fragments were amino acid sequenced and this permitted, in combination with the tryptic peptides (Carlsberg Res. Commun. 48, 517-527 (1983)), identification of 574 of the 614 amino acid residues in Gl. Sequencing of glucoamylase Gl cDNA, constructed from A. niger total mRNA, enabled deduction of the sequence of the remaining 40 amino acid residues localized to 6 short stretches. From the alignment of the fragments the complete primary structure of the enzyme was established. The amino acid sequence corresponds to a molecular weight of the polypeptide moiety of 65,424. Including both hexosamine and neutral carbohydrate contents the molecular weight of the present sample of G1 was calculated to be about 82,000.The majority of the carbohydrate of Gl is found in a highly glycosylated region of 70 amino acid residues which comprises about 35 O-glycosyl serine and threonine residues. This region ends approximately 100 residues from the C-terminus of the enzyme. Two N-glycosylated positions were found in the central part of the polypeptide chain. The molecule contains 9 half-cystine residues. No homology is apparent between the sequence of glycoamylase and various at-amylases.Abbreviations: BNPS-skatole = 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine; ca = citraconyl; Con A = concanavalin A; DFP = diisopropylfluorophosphate; DPCC = diphenylcarbamyl chloride; EDTA = ethylenediaminetetraacetic acid, disodium salt; Hepes = N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid; Nemac = N-ethylmorpholine acetate; HPLC = high pressure liquid chromatography; PTH = phenylthiohydantoin; SDS-PAGE = polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate; 2-pe = 2-pyridylethyl; G I designates the larger and G2 the smaller of the forms ofglucoamylase from A. niger (44).

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Section: Construction and Cloning Of Doublestranded Glucoamylase G1 Cdnamentioning

confidence: 99%

The complete amino acid sequence of the glycoprotein, glucoamylase G1, from Aspergillus niger

Svensson

Larsen

Svendsen

et al. 1983

Carlsberg Res. Commun.

189

158

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“…Plasma desorption mass spectra were obtained from 90-120-pmol aliquots and 0.2-0.4X10' primary ions were recorded. Numbers in brackets were calculated from the cDNA-derived sequence of human progastrin [15]. The starting material contained 160 nmol antiserum 88235 immunoreactivity, as corrected for the lower reactivity of peptides extended N-terminally to Asp20.…”

Section: Discussionmentioning

confidence: 99%

“…Consequently, gastrin peptides are all derived from a single progastrin molecule, which in mammals has the same overall structure and size [15,[17][18][19][20][21][22]. A decisive feature of the gastrins is the unusually well-defined active site (-Trp-MetAsp-Phe-NH,) that constitutes the C-terminus of all bioactive gastrins.…”

Section: Definition Of the Gastrin Systemmentioning

confidence: 99%

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Identification of gastrin component I as gastrin‐71

Rehfeld

Johnsen

1994

European Journal of Biochemistry

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Gastrin component I is the largest hormonally active form of gastrin. In order to determine its structure, we isolated progastrin-derived peptides from normal human antral tissue. A radioimmunoassay specific for sequence 20-25 of human progastrin was developed to monitor the purifications. After four or five steps of reverse-phase chromatography, the peptides were pure and could be identified by a combination of microsequence, amino acid and mass spectral analysis as well as by a library of sequence-specific immunoassays. In addition to intact progastrin 1-80, fragments 1-71, 1-35, 6-35, 20-35, and 20-36 of progastrin were identified. Only the 71-amino-acid peptide contained at its C-terminus the a-amidated bioactive site (Trp-Met-Asp-Phe-NH,). This unoheptacontapeptide amide (gastrin-71) corresponds to component I and is the largest possible bioactive product of progastrin. Its structure shows that progastrin is used in its entirety for biosynthesis of active peptides. The occurrence of fragments 6-35,20-35, and 20-36 demonstrate that antral progastrin is partially cleaved at two monobasic sites (Arg5 and Argl9) in addition to processing at the three C-terminal dibasic sites. The results show that both the N-and C-terminal parts of antral progastrin undergo extensive processing. The results also suggest that progastrin may follow two different processing pathways of which the less trafficked releases gastrin-71.Biologically active peptides (hormones, neuropeptides, cytokines, and growth factors) are characterized by high specific bioactivity at low concentrations. The peptides are synthesized from long precursors, and achieve their unique bioactivity only after elaborate and precise precursor processing. During the last decade much information about tissue-specific precursor processing has accumulated (for review, see

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“…The construction and in vitro transcription of the human ppENK-A and rat ppCCK DNA templates were recently described 112,131. The human gastrin DNA template was constructed by subcloning a 280 bp HindIIl/ExonIII-DNA fragment [23] into the Hind111 site of pGEM-3 vector (Promega Inc.). The @-Actin template was obtained by subcloning a -1150 bp &t-DNA fragment [24] into vector pGEM-3.…”

Section: Hybridization Dna Templates and Crna Probesmentioning

confidence: 99%

Expression of the cholecystokinin gene in a human (small‐cell) lung carcinoma cell‐line

Geijer¹,

Folkesson

Rehfeld

et al. 1990

FEBS Letters

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Expression of the cholecystokinin (CCK), gastrin and enkephalin A genes were studied by Northern blot analysis and a library of sequence-specific radioimmunoassays in human cell lines. The human small-cell lung carcinoma line (SCLC) U-1690 expressed moderate levels of CCK mRNA as compared to the human neuroepithelioma cell line SK-N-MC. Neither gastrin nor (pro)enkephalin A mRNAs were detectable in the U-1690 cell line. In contrast, the SCLC-line H-69 expressed Enk A but no CCK mRNA. The radioimmunoassays showed that the CCK mRNA transcript in the SCLC line U-1690 also is translated, and that preproCCK is processed into bioactive, carboxyamidated CCK peptides. Thus, the human small cell carcinoma cell line U-1690 is a useful model for studies of cell-specific CCK gene expression.

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Molecular cloning of human gastrin cDNA: evidence for evolution of gastrin by gene duplication.

Cited by 174 publications

References 31 publications

The complete amino acid sequence of the glycoprotein, glucoamylase G1, from Aspergillus niger

The complete amino acid sequence of the glycoprotein, glucoamylase G1, from Aspergillus niger

Identification of gastrin component I as gastrin‐71

Expression of the cholecystokinin gene in a human (small‐cell) lung carcinoma cell‐line

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