RESUMO
Anglerfish (Lophius americanus) insulin complementary DNA was cloned in bacterial plasmids, and its sequence was determined. Fish insulin messenger RNA is larger (1.5 times) than the messenger RNA encoding mammalian (rat and human) insulin, in part because of a larger C peptide (an additional six amino acids or 18 nucleotides in length) but mainly because of increases in the 5' and 3' untranslated regions. Comparison of the fish, rat, and human insulin messenger RNA (from the complementary DNA) reveals that, in addition to the regions coding for the A and B peptides, sequence conservation is limited to a segment within the 5' untranslated region which may be involved in ribosomal binding, two small segments of the signal peptide, and two stretches of sequence in the 3' untranslated region.
Assuntos
Evolução Biológica , Peixes/genética , Insulina/genética , RNA Mensageiro/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , Códon , Conformação de Ácido Nucleico , Proinsulina/genética , Biossíntese de Proteínas , Precursores de Proteínas/genéticaRESUMO
It has been proposed that the endocrine cells of the digestive tract derive from the neuroectoderm (neural crest). To test this hypothesis we removed the entire ectoderm, the precursor of the neural crest, of embryonic rats prior to the formation of the neural crest and cultured the mesoendoderm for 11 days. In every case where a pancreas developed, insulin was detected or B cells were observed. Thus, a neural crest origin for these cells is eiliminated.
Assuntos
Ectoderma/fisiologia , Insulina/biossíntese , Ilhotas Pancreáticas/embriologia , Sistema Nervoso/embriologia , Aminas/metabolismo , Animais , Diferenciação Celular , Sistema Digestório/embriologia , Ectoderma/metabolismo , Glândulas Endócrinas/embriologia , Hormônios Gastrointestinais/biossíntese , RatosRESUMO
The ontogenesis of immunoreactive somatostatin in the embryonic and fetal rat pancreas has been measured by radioimmunoassay following acid extraction. Somatostatin (GIF) is detectable at 14 days gestation at a concentration of 1.6 X 10(-3) ng/pancreas. At term the content is 3.8 ng/pancreas, by 2 days neonatally, 8.3 ng/pancreas, and in the adult rat, 71 ng/pancreas through the concentration (expressed per microgram DNA) is constant from 14-19 days of gestation and reaches a level characteristic of the fully differentiated pancreas by birth. The detection of GIF in cultured pancreatic explants in the absence of innervation indicates that synthesis can occur independent of neural influence.
Assuntos
Pâncreas/embriologia , Somatostatina/metabolismo , Animais , Animais Recém-Nascidos , Denervação , Epitélio/metabolismo , Feminino , Feto/metabolismo , Idade Gestacional , Masculino , Pâncreas/inervação , Gravidez , Radioimunoensaio , RatosRESUMO
Endocrine B cells differentiate normally in embryonic rat pancreatic rudiments cultured in vitro. The specific concentration of immunoreactive insulin based on total protein increases by about 1000-fold during the developmental period, corresponding to days 13--20 of gestation. The rate of (pro)insulin synthesis, measured from the level of radioactive leucine incorporated into insulin, quantitatively accounts for the insulin accumulated during this period. In addition, the relative incorporation of leucine into proinsulin compared to insulin is constant during development and is similar to that found in the B cells of adult islets. Thus, there appears to be no significant change in the rate of conversion of proinsulin to insulin during B cell differentiation.
Assuntos
Insulina/biossíntese , Ilhotas Pancreáticas/citologia , Pâncreas/embriologia , Proinsulina/biossíntese , Animais , Diferenciação Celular , Técnicas de Cultura de Órgãos , Pâncreas/metabolismo , Biossíntese de Proteínas , RatosRESUMO
RNA has been isolated from a human pancreatic somatostatinoma and used to prepare a cDNA library. After prescreening, clones containing somatostatin I sequences were identified by hybridization with an anglerfish somatostatin I-cloned cDNA probe. From the nucleotide sequence of two of these clones, we have deduced an essentially full-length mRNA sequence, including the preprosomatostatin coding region, 105 nucleotides from the 5' untranslated region and the complete 150-nucleotide 3' untranslated region. The coding region predicts a 116-amino acid precursor protein (Mr, 12.727) that contains somatostatin-14 and -28 at its COOH terminus. The predicted amino acid sequence of human somatostatin-28 is identical to that of somatostatin-28 isolated from the porcine and ovine species. A comparison of the amino acid sequences of human and anglerfish preprosomatostatin I indicated that the COOH-terminal region encoding somatostatin-14 and the adjacent 6 amino acids are highly conserved, whereas the remainder of the molecule, including the signal peptide region, is more divergent. However, many of the amino acid differences found in the pro region of the human and anglerfish proteins are conservative changes. This suggests that the propeptides have a similar secondary structure, which in turn may imply a biological function for this region of the molecule.
Assuntos
DNA/genética , Somatostatina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Recombinante , Peixes , Humanos , Neoplasias Pancreáticas/análise , Plasmídeos , Precursores de Proteínas/genética , RNA Mensageiro/genética , Somatostatina/análogos & derivados , Somatostatinoma/análiseRESUMO
Glucagon and insulin are first detectable at the onset of rat pancreas organogenesis. Initially, the specific activity of glucagon is approximately 100-fold higher than that of insulin. At this early stage, endocrine storage granules, similar to alpha granules, are identifiable in electron micrographs. The granule characteristics, as well as the relative hormone levels, suggest that the early population of differentiated endocrine cells is in fact composed of glucagon-producing (A) cells. This high level of glucagon is present in the embryo much earlier than the metabolic processes thought to be controlled by this hormone. Moreover, glucagon-producing cells may be the first endocrine cells to differentiate. Other known endocrine products accumulate later, during the terminal stages of organogenesis. These observations suggest that glucagon may have a regulatory function in early embryogenesis.
Assuntos
Glucagon/biossíntese , Pâncreas/embriologia , Animais , Diferenciação Celular , Grânulos Citoplasmáticos , Idade Gestacional , Glucagon/análise , Complexo de Golgi , Imunoensaio , Insulina/análise , Insulina/biossíntese , Microscopia Eletrônica , Pâncreas/citologia , Pâncreas/metabolismo , RatosRESUMO
Portions of at least nine distinct rat amylase genes or pseudogenes have been isolated. Cloned rat genomic DNA fragments containing complete or major portions of seven of these have been examined by heteroduplex analysis and fall within two separate groups based on their degree of homology. Four gene sequences comprising one of these groups are closely related to pancreatic amylase mRNA. The other group shows significant nonhomology to both pancreatic and parotid amylase cDNAs and may represent an additional gene type(s). All of the cloned amylase gene sequences are found in rat genomic DNA. Additional amylase sequences which have not yet been cloned are also detected. Comparison of DNA from individual Sprague-Dawley rats by Southern blotting techniques indicates allelic variation at multiple amylase loci.
Assuntos
Amilases/genética , Genes , Animais , Sequência de Bases , Clonagem Molecular , Enzimas de Restrição do DNA/metabolismo , Desoxirribonuclease EcoRI , Microscopia Eletrônica , Ratos , Ratos EndogâmicosRESUMO
The sequences of two cloned rat pancreatic amylase cDNAs comprising 95% of the mRNA sequence are reported. Analysis of cloned rat genomic DNA fragments using cloned cDNA probes indicates that the rat genome contains multiple closely related amylase genes in which the cDNA sequences are distributed within a region 9 kilobases in length and are interrupted by at least seven intervening sequences.
Assuntos
Amilases/genética , Pâncreas/enzimologia , RNA Mensageiro/genética , alfa-Amilases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Genes , Ligação Genética , Ratos , Relação Estrutura-AtividadeRESUMO
The human insulin gene contains two intervening sequences, one is within the region transcribed into the 5'-untranslated segment of the mRNA and the other interrupts the C-peptide encoding region. A comparison of the human with the rat insulin genes indicates potential regulatory regions in the DNA segment preceding the gene and suggests that the ancestral form of the insulin gene had two intervening sequences.
Assuntos
Genes , Insulina/genética , Alelos , Animais , Sequência de Bases , Códon , Enzimas de Restrição do DNA , Variação Genética , Humanos , Óperon , RNA Mensageiro/genética , Ratos/genéticaRESUMO
The mRNA sequences for two rat pancreatic elastolytic enzymes have been cloned by recombinant DNA technology and their nucleotide sequences determined. Rat elastase I mRNA is 1113 nucleotides in length, plus a poly(A) tail, and encodes a preproelastase of 266 amino acids. The amino acid sequence of the predicted active form of rat elastase I is 84% homologous to porcine elastase 1. Key amino acid residues involved in determining substrate specificity of porcine elastase 1 are retained in the rat enzyme. The activation peptide of the zymogen does not appear related to that of other mammalian pancreatic serine proteases. The mRNA for elastase I is localized in the rough endoplasmic reticulum of acinar cells, as expected for the site of synthesis of an exocrine secretory enzyme. Rat elastase II mRNA is 910 nucleotides in length, plus a poly(A) tail, and encodes a preproenzyme of 271 amino acids. The amino acid sequence is more closely related to porcine elastase 1 (58% sequence identity) than to the other pancreatic serine proteases (33-39% sequence identity). Predictions of substrate preference based upon key amino acid residues that define the substrate binding cleft are consistent with the broad specificity observed for mammalian pancreatic elastase 2. The activation peptide is similar to that of the chymotrypsinogens and retains an N-terminal cysteine available to form a disulfide link to an internal conserved cysteine residue.
Assuntos
Clonagem Molecular , Precursores Enzimáticos/genética , Pâncreas/enzimologia , Elastase Pancreática/genética , RNA Mensageiro , Sequência de Aminoácidos , Animais , Sequência de Bases , RatosRESUMO
Via recombinant DNA technology the mRNA sequence of pancreatic amylase has been cloned and its nucleotide sequence has been determined. The cloned sequence represents 96% of the total length of amylase mRNA; missing are an estimated 75 +/- 30 nucleotides from the 5' end. The amino acid sequence of rat pancreatic amylase was deduced solely from the nucleotide sequence of the mRNA. Unlike other eukaryotic mRNAs, the amylase mRNA has short 5' and 5' untranslated regions, suggesting that long untranslated regions of eukaryotic mRNAs either do not contain extensive functional sequences or that these sequences are incorporated within the amino acid coding region of amylase mRNA. The cloned amylase mRNA sequence was radiolabeled and used as a probe for in situ hybridization. These experiments demonstrate that amylase mRNA is present in all acinar cells but not in other pancreatic cell types. Using the cloned amylase mRNA sequences as a hybridization probe, three nonoverlapping genomic DNA fragments containing amylase gene sequences were isolated. From the similar sequence organization of the three amylase genes visualized by DNA heteroduplex mapping, a consensus structure of a rat amylase gene is proposed. It is an extended gene structure 10 kilobase pairs in length containing the 1547 base pairs of the cloned mRNA coding sequence interrupted by seven intervening sequences ranging from 400-2000 base pairs long. Thus, in nuclear DNA the amylase mRNA coding sequence is disrupted into at least eight segments from 150-300 base pairs long.
Assuntos
Amilases/genética , Genes , Pâncreas/enzimologia , RNA Mensageiro , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Enzimas de Restrição do DNA , DNA Recombinante , Regulação da Expressão Gênica , Código Genético , Hibridização de Ácido Nucleico , Biossíntese de Proteínas , RatosRESUMO
Rat carboxypeptidase A cDNA clones have been isolated from a cDNA library prepared from pancreatic mRNA. An almost complete mRNA sequence has been deduced that predicts a polypeptide having 78% amino acid sequence homology with bovine carboxypeptidase A. The amino acid sequence of the activation and signal peptides of the carboxypeptidase A precursor were inferred from the nucleotide sequence. The cDNA was used as a probe to identify DNA fragments containing carboxypeptidase A sequences in a bacteriophage lambda library of rat genomic DNA. Heteroduplexes revealed that the DNA coding sequence occupies 5.5 kilobases and is interrupted by nine intervening sequences. The nucleotide sequence of the 5' end of the gene and the adjacent flanking region provides information on the site of initiation of transcription and the putative control regions. There is no evident relationship between the localization of intervening sequences in the gene and functional/structural domains of the protein.