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1.
J Pharmacol Exp Ther ; 296(3): 1058-66, 2001 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-11181941

RESUMO

Histamine exerts its numerous physiological functions through interaction with G protein-coupled receptors. Three such receptors have been defined at both the pharmacological and molecular level, while pharmacological evidence hints at the existence of further subtypes. We report here the cloning and characterization of a fourth histamine receptor subtype. Initially discovered in an expressed-sequence tag database, the full coding sequence (SP9144) was subsequently identified in chromosome 18 genomic sequence. This virtual coding sequence exhibited highest homology to the H(3) histamine receptor and was used to generate a full-length clone by polymerase chain reaction (PCR). The distribution of mRNA encoding SP9144 was restricted to cells of the immune system as determined by quantitative PCR. HEK-293 cells transiently transfected with SP9144 and a chimeric G protein alpha-subunit (Galpha(q/i1,2)) exhibited increases in intracellular [Ca(2+)] in response to histamine but not other biogenic amines. SP9144-transfected cells exhibited saturable, specific, high-affinity binding of [(3)H]histamine, which was potently inhibited by H(3) receptor-selective compounds. The rank order and potency of these compounds at SP9144 differed from the rank order at the H(3) receptor. Although SP9144 apparently coupled to Galpha(i), HEK-293 cells stably transfected with SP9144 did not exhibit histamine-mediated inhibition of forskolin-stimulated cAMP levels. However, both [(35)S]GTPgammaS binding and phosphorylation of mitogen-activated protein kinase were stimulated by histamine via SP9144 activation. In both of these assays, SP9144 exhibited evidence of constitutive activation. Taken together, these data demonstrate that SP9144 is a unique, fourth histamine receptor subtype.


Assuntos
Histamina/metabolismo , Receptores Histamínicos H3/genética , Receptores Histamínicos/genética , Tioureia/análogos & derivados , Sequência de Aminoácidos , Células Cultivadas , Clonagem Molecular , Agonistas dos Receptores Histamínicos/farmacologia , Humanos , Imidazóis/farmacologia , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ensaio Radioligante , Receptores Histamínicos/efeitos dos fármacos , Receptores Histamínicos H3/efeitos dos fármacos , Homologia de Sequência de Aminoácidos , Tioureia/farmacologia , Distribuição Tecidual , Transfecção
2.
J Biol Chem ; 276(11): 8608-15, 2001 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-11104774

RESUMO

P2Y receptors are a class of G protein-coupled receptors activated primarily by ATP, UTP, and UDP. Five mammalian P2Y receptors have been cloned so far including P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11. P2Y1, P2Y2, and P2Y6 couple to the activation of phospholipase C, whereas P2Y4 and P2Y11 couple to the activation of both phospholipase C and the adenylyl cyclase pathways. Additional ADP receptors linked to Galpha(i) have been described but have not yet been cloned. SP1999 is an orphan G protein-coupled receptor, which is highly expressed in brain, spinal cord, and blood platelets. In the present study, we demonstrate that SP1999 is a Galpha(i)-coupled receptor that is potently activated by ADP. In an effort to identify ligands for SP1999, fractionated rat spinal cord extracts were assayed for Ca(2+) mobilization activity against Chinese hamster ovary cells transiently transfected with SP1999 and chimeric Galpha subunits (Galpha(q/i)). A substance that selectively activated SP1999-transfected cells was identified and purified through a series of chromatographic steps. Mass spectral analysis of the purified material definitively identified it as ADP. ADP was subsequently shown to inhibit forskolin-stimulated adenylyl cyclase activity through selective activation of SP1999 with an EC(50) of 60 nM. Other nucleotides were able to activate SP1999 with a rank order of potency 2-MeS-ATP = 2-MeS-ADP > ADP = adenosine 5'-O-2-(thio)diphosphate > 2-Cl-ATP > adenosine 5'-O-(thiotriphosphate). Thus, SP1999 is a novel, Galpha(i)-linked receptor for ADP.


Assuntos
Difosfato de Adenosina/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Receptores Purinérgicos P2/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Clonagem Molecular , Cricetinae , DNA Complementar/química , Perfilação da Expressão Gênica , Humanos , Ligantes , Camundongos , Dados de Sequência Molecular , Ratos , Receptores Purinérgicos P2Y1
3.
Regul Pept ; 75-76: 45-53, 1998 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-9802393

RESUMO

NPY is a 36-amino acid peptide which exerts its physiological effects through the activation of a family of G-protein coupled receptors. In vivo and in vitro characterization of the recently cloned rat Y5 receptor suggests that it is a primary mediator of NPY-induced feeding (Gerald et al., Nature 1996;382:168-171). We now report the molecular cloning and pharmacological characterization of the human, dog and mouse homologs of the Y5 receptor. With the exception of a 21 amino acid repeat in the amino terminus of the mouse Y5 receptor, the sequence of the four species homologs appear to be highly conserved, with 88% to 97% amino acid identities between any two species. Similarly, the pharmacological profiles of the four species homologs as determined in porcine 125I-PYY binding assays show a great deal of conservation, with the following rank order of affinity: human or porcine NPY, PYY, [Leu31,Pro34]NPY, NPY(2-36), human PP > human [D-Trp32]NPY > rat PP, C2-NPY. Northern blot analysis reveals that the Y5 receptor is widely distributed in the human brain, with the strongest signals detected in the cortex, putamen and caudate nucleus. The chromosomal localization of the human Y5 receptor, previously shown to be overlapping and in the opposite orientation to the Y1 receptor, is determined to be 4q31, the same locus as previously demonstrated for the human Y1 receptor (Herzog et al., J Biol Chem 1993;268:6703-6707), suggesting that these receptors may be coregulated. These Y5 species homologs along with corresponding animal models may be useful in the search for novel therapeutics in the treatment of obesity and related feeding disorders.


Assuntos
Receptores de Neuropeptídeo Y/genética , Receptores de Neuropeptídeo Y/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Cromossomos Humanos Par 4/genética , Clonagem Molecular , Cães , Humanos , Hibridização in Situ Fluorescente , Cinética , Camundongos , Dados de Sequência Molecular , Neuropeptídeo Y/metabolismo , Polipeptídeo Pancreático/metabolismo , Peptídeo YY/metabolismo , Filogenia , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Transfecção
4.
Nature ; 382(6587): 168-71, 1996 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-8700207

RESUMO

Neuropeptide Y (NPY) is a powerful stimulant of food intake and is proposed to activate a hypothalamic 'feeding' receptor distinct from previously cloned Y-type receptors. This receptor was first suggested to explain a feeding response to NPY and related peptides, including NPY2-36, that differed from their activities at the Y1 receptor. Here we report the expression cloning of a novel Y-type receptor from rat hypothalamus, which we name Y5. The complementary DNA encodes a 456-amino-acid protein with less than 35% overall identity to known Y-type receptors. The messenger RNA is found primarily in the central nervous system, including the paraventricular nucleus of the hypothalamus. The extent to which selected peptides can inhibit adenylate cyclase through the Y5 receptor and stimulate food intake in rats correspond well. Our data support the idea that the Y5 receptor is the postulated 'feeding' receptor, and may provide a new method for the study and treatment of obesity and eating disorders.


Assuntos
Comportamento Alimentar/fisiologia , Neuropeptídeo Y/fisiologia , Receptores de Neuropeptídeo Y/fisiologia , Sequência de Aminoácidos , Animais , Bovinos , Linhagem Celular , Clonagem Molecular , Humanos , Hipotálamo/fisiologia , Masculino , Dados de Sequência Molecular , Peptídeos/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de Neuropeptídeo Y/efeitos dos fármacos , Receptores de Neuropeptídeo Y/genética , Suínos , Transfecção
5.
Mol Microbiol ; 19(6): 1225-39, 1996 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-8730865

RESUMO

The secondary structure and sequences influencing the expression and selection of the AUG initiator codon in the yeast Saccharomyces cerevisiae were investigated with two fused genes, which were composed of either the CYC7 or CYC1 leader regions, respectively, linked to the lacZ coding region. In addition, the strains contained the upf1-delta disruption, which stabilized mRNAs that had premature termination codons, resulting in wild-type levels. The following major conclusions were reached by measuring beta-galactosidase activities in yeast strains having integrated single copies of the fused genes with various alterations in the 89 and 38 nucleotide-long untranslated CYC7 and CYC1 leader regions, respectively. The leader region adjacent to the AUG initiator codon was dispensable, but the nucleotide preceding the AUG initiator at position -3 modified the efficiency of translation by less than twofold, exhibiting an order of preference A > G > C > U. Upstream out-of-frame AUG triplets diminished initiation at the normal site, from essentially complete inhibition to approximately 50% inhibition, depending on the position of the upstream AUG triplet and on the context (-3 position nucleotides) of the two AUG triplets. In this regard, complete inhibition occurred when the upstream and downstream AUG triplets were closer together, and when the upstream and downstream AUG triplets had, respectively, optimal and suboptimal contexts. Thus, leaky scanning occurs in yeast, similar to its occurrence in higher eukaryotes. In contrast, termination codons between two AUG triplets causes reinitiation at the downstream AUG in higher eukaryotes, but not generally in yeast. Our results and the results of others with GCN4 mRNA and its derivatives indicate that reinitiation is not a general phenomenon in yeast, and that special sequences are required.


Assuntos
Códon de Iniciação/genética , RNA Fúngico/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Sequência de Bases , DNA Fúngico/genética , Genes Fúngicos , Vetores Genéticos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Plasmídeos/genética , Biossíntese de Proteínas , RNA Fúngico/química , RNA Mensageiro/química
6.
EMBO J ; 14(12): 2806-15, 1995 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-7796807

RESUMO

Molecular cloning efforts have provided primary amino acid sequence and signal transduction data for a large collection of serotonin receptor subtypes. These include five 5-HT1-like receptors, three 5-HT2 receptors, one 5-HT3 receptor, two 5-HT5 receptors, one 5-HT6 receptor and one 5-HT7 receptor. Molecular biological information on the 5-HT4 receptor is notably absent from this list. We now report the cloning of the pharmacologically defined 5-HT4 receptor. Using degenerate oligonucleotide primers, we identified a rat brain PCR fragment which encoded a '5-HT receptor-like' amino acid sequence. The corresponding full length cDNA was isolated from a rat brain cDNA library. Transiently expressed in COS-7 cells, this receptor stimulates adenylyl cyclase activity and is sensitive to the benzamide derivative cisapride. The response is also blocked by ICS-205930. Interestingly, we isolated two splice variants of the receptor, 5-HT4L and 5-HT4S, differing in the length and sequence of their C-termini. In rat brain, the 5-HT4S transcripts are restricted to the striatum, but the 5-HT4L transcripts are expressed throughout the brain, except in the cerebellum where it was barely detectable. In peripheral tissues, differential expression was also observed in the atrium of the heart where only the 5-HT4S isoform was detectable.


Assuntos
RNA Mensageiro/análise , Receptores de Serotonina/genética , Adenilil Ciclases/metabolismo , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Sequência de Bases , Química Encefálica , Linhagem Celular , Cisaprida , Clonagem Molecular , DNA Complementar/genética , Expressão Gênica , Átrios do Coração/química , Indóis/farmacologia , Dados de Sequência Molecular , Especificidade de Órgãos , Piperidinas/farmacologia , Splicing de RNA , Ratos , Receptores de Serotonina/química , Receptores de Serotonina/metabolismo , Receptores 5-HT4 de Serotonina , Análise de Sequência de DNA , Serotonina/metabolismo , Antagonistas da Serotonina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Tropizetrona
7.
Mol Pharmacol ; 46(3): 414-22, 1994 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-7935320

RESUMO

The cDNA for the rat alpha 1c-adrenergic receptor (AR) has been cloned using a probe derived from the bovine alpha 1c-AR sequence. Clone rB7a has a 2.6-kilobase insert with a 1390-base pair open reading frame and encodes a receptor of 466 amino acids. The cloned receptor has 91% amino acid identity with the bovine alpha 1c-AR. The rat alpha 1c-AR mRNA was detected in tissues known to be enriched for the alpha 1A-AR subtype, including vas deferens, heart, kidney, and hippocampus. Rat alpha 1c-AR mRNA was absent from liver and spleen when assayed by Northern blot analyses and RNase protection assays. In COS-7 cells transfected with cDNAs encoding the three rat alpha 1-ARs, WB-4101 and benoxathian had similar binding affinities for the alpha 1a/d-AR and the alpha 1c-AR and 10-fold lower affinities for the alpha 1b-AR. The affinity of 5-methylurapidil was found to be 10- and 30-fold higher at the alpha 1c-AR than at the alpha 1a/d- and alpha 1b-ARs, respectively. (S)-(+)-Niguldipine was found to have high affinity for the rat alpha 1c-AR, with 42- and 22-fold lower affinity at the alpha 1a/d- and alpha 1b-ARs, respectively. Treatment of intact transfected COS-7 cells with chlorethylclonidine resulted in the inactivation of 19% of the alpha 1c-ARs, in contrast to 72% and 85% inactivation of the alpha 1a/d- and alpha 1b-ARs, respectively. Similarly to the other two alpha 1-ARs, the rat alpha 1c-AR is coupled to the activation of phospholipase C. Our data suggest that the rat alpha 1c-AR cDNA encodes an alpha 1-AR with the pharmacological properties previously defined for the alpha 1A subtype found in tissues.


Assuntos
Agonistas alfa-Adrenérgicos/metabolismo , Antagonistas Adrenérgicos alfa/metabolismo , Receptores Adrenérgicos alfa/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Northern Blotting , Bovinos , Linhagem Celular , Clonidina/análogos & derivados , Clonidina/farmacologia , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Ativação Enzimática/efeitos dos fármacos , Expressão Gênica/genética , Glicosilação , Humanos , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Fases de Leitura Aberta , Fosforilação , RNA Mensageiro/análise , Ratos , Receptores Adrenérgicos alfa/classificação , Receptores Adrenérgicos alfa/efeitos dos fármacos , Receptores Adrenérgicos alfa/genética , Ribonucleases/química , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fosfolipases Tipo C/metabolismo
8.
Mol Cell Biol ; 14(6): 3752-62, 1994 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-8196618

RESUMO

PRL-1 is a particularly interesting immediate-early gene because it is induced in mitogen-stimulated cells and regenerating liver but is constitutively expressed in insulin-treated rat H35 hepatoma cells, which otherwise show normal regulation of immediate-early genes. PRL-1 is expressed throughout the course of hepatic regeneration, and its expression is elevated in a number of tumor cell lines. Sequence analysis reveals that PRL-1 encodes a 20-kDa protein with an eight-amino-acid consensus protein tyrosine phosphatase (PTPase) active site. PRL-1 is able to dephosphorylate phosphotyrosine substrates, and mutation of the active-site cysteine residue abolishes this activity. As PRL-1 has no homology to other PTPases outside the active site, it is a new type of PTPase. PRL-1 is located primarily in the cell nucleus. Stably transfected cells which overexpress PRL-1 demonstrate altered cellular growth and morphology and a transformed phenotype. It appears that PRL-1 is important in normal cellular growth control and could contribute to the tumorigenicity of some cancer cells.


Assuntos
Divisão Celular/fisiologia , Núcleo Celular/enzimologia , Genes Precoces , Proteínas Imediatamente Precoces/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Southern Blotting , Proteínas de Ciclo Celular , Linhagem Celular , Sequência Consenso , Primers do DNA , Humanos , Cinética , Neoplasias Hepáticas Experimentais , Proteínas de Membrana , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas de Neoplasias , Especificidade de Órgãos , Reação em Cadeia da Polimerase , Proteínas Tirosina Fosfatases/biossíntese , Ratos , Homologia de Sequência de Aminoácidos , Fatores de Tempo , Células Tumorais Cultivadas
9.
J Biol Chem ; 268(12): 8855-61, 1993 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-8473329

RESUMO

Liver regeneration following partial hepatectomy provides one of the few systems for analysis of mitogenesis in the fully developed, intact animal. Immediate-early growth response genes, induced in the absence of prior protein synthesis, play an important regulatory role in the regenerative process. During screening of a subtracted cDNA library of immediate-early genes induced during liver regeneration, a novel member of the thyroid/steroid receptor superfamily, RNR-1 (regenerating liver nuclear receptor), was identified. This gene is not expressed in quiescent liver but is rapidly induced following partial hepatectomy and is specific to hepatic growth as it is not induced in other mitogen-treated cells. RNR-1 is also expressed in brain. A full-length cDNA clone of RNR-1 encodes a 66-kDa, 597-amino acid protein as verified by in vitro translation in reticulocyte lysate. RNR-1 is highly homologous to r-NGFI-B/m-Nur77 particularly in the DNA binding (94%) and putative ligand binding (59%) domains. Using a mobility shift assay, we have shown that RNR-1 specifically binds to the NGFI-B DNA half-site and forms a complex very similar in size to the Nur77 complex, suggesting that RNR-1 also may bind as a monomer. Consistent with this finding, the A box region important in mediating half-site binding is 100% conserved between r-NGFI-B/m-Nur77. Both RNR-1 and Nur77 strongly transactivate a reporter driven by a consensus r-NGFI-B/Nur77 binding site, and their effect together is additive. As both the RNR-1 and r-NGFI/m-nur77 genes are induced during liver regeneration, it is very possible that RNR-1 acts concomitantly with r-NGFI/m-Nur77 in regulating the expression of delayed-early genes during liver regeneration.


Assuntos
Proteínas de Ligação a DNA/genética , Regeneração Hepática/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Células 3T3 , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Northern Blotting , Núcleo Celular/metabolismo , Células Cultivadas , DNA , Proteínas de Ligação a DNA/metabolismo , Feminino , Fase G1/genética , Camundongos , Dados de Sequência Molecular , Família Multigênica , Proteínas Nucleares/metabolismo , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares , Biossíntese de Proteínas , Ratos , Ratos Endogâmicos F344 , Receptores Citoplasmáticos e Nucleares , Receptores de Esteroides , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/metabolismo
10.
Mol Cell Biol ; 11(3): 1393-401, 1991 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-1705004

RESUMO

The liver is an epithelioid organ that can regenerate following partial hepatectomy. Although it is composed mainly of hepatocytes, it has a complex, multicellular architecture, implying that intercellular communications must exist during regeneration. As in other mitogen-stimulated cells, immediate-early growth response genes induced in the absence of prior protein synthesis are likely to play an important regulatory role in the regenerative process. Through differential screening of regenerating liver cDNA libraries, we found that one of the most highly expressed immediate-early genes in liver regeneration encodes the rat homolog of the low-molecular-weight insulinlike growth factor (IGF)-binding protein (IGFBP-1). This protein has been implicated in enhancing the mitogenic effect of IGF on tissues. IGFBP-1 gene induction is transcriptionally mediated and specific to regenerating liver, as the gene is not expressed in mitogen-stimulated fibroblasts. IGFBP-1 expression has been shown to increase under low-insulin conditions such as diabetes, and the complex regulation of expression is indicated by our finding that insulin treatment of H35 rat hepatoma cells, which induces proliferation, also causes a rapid decrease in transcription and expression of the IGFBP-1 gene. Of note, IGFBP-1 mRNA is abundant in fetal rat liver, implying that it participates in normal liver growth and development. Although regenerating liver cells continue to produce IGF-I, we did not detect IGF-I receptor mRNA during the first 24 h after hepatectomy. However, some IGFBPs may act to enhance the activity of IGF-I independently of IGF-I receptors. Thus, IGF-1 and IGFBPs may interact with hepatocytes or nonparenchymal liver cells, through either IGF-I or novel receptors. In this way, IGFBP-I and IGF-I could act in a paracrine and/or autocrine fashion in maintaining normal liver architecture during regeneration.


Assuntos
Proteínas de Transporte/genética , Regeneração Hepática , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , DNA/genética , Expressão Gênica , Insulina/farmacologia , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina , Fator de Crescimento Insulin-Like I/genética , Fígado/embriologia , Fígado/fisiologia , Dados de Sequência Molecular , Ratos , Receptores de Superfície Celular/genética , Receptores de Somatomedina
11.
Mol Cell Biol ; 11(1): 381-90, 1991 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-1986233

RESUMO

Liver regeneration provides a unique system for analysis of mitogenesis in intact, fully developed animals. Cellular immediate-early genes likely play an important role in cell cycle regulation and have been extensively studied in mitogen-stimulated fibroblasts lymphocytes but not in liver. We have begun to characterize the immediate-early growth response genes of mitogen-stimulated liver cells, specifically, regenerating liver and insulin-stimulated Reuber H-35 hepatoma cells, and to address differences in growth response between different cell types. Through subtraction and differential screening of cDNA libraries from regenerating liver and insulin-treated H-35 cells, we have extensively characterized 341 differentially expressed clones and identified 52 immediate-early genes. These genes have been partially sequenced and subjected to Northern (RNA) blot analysis, and 41 appear to be novel. Surprisingly, two-thirds of these genes are also expressed in BALB/c 3T3 cells, but only 10 were identified in previous studies of 3T3 cells, and of these, 6 include well-known genes like jun and fos, and only 4 are novel. Approximately one-third of the immediate-early genes identified in mitogen-stimulated liver cells or serum-stimulated NIH 3T3 cells are expressed in a tissue-specific fashion, indicating that cell type-specific regulation of the proliferative response occurs during the immediate-early period. Our findings indicate that the immediate-early response is unusually complex for the first step in a regulatory cascade, suggesting that multiple pathways must be activated. The abundance of immediate-early genes and the highly varied pattern of their expression in different cell types suggest that the tissue specificity of the proliferative response arises from the particular set of these genes expressed in a given tissue.


Assuntos
Ciclo Celular , Expressão Gênica , Insulina/farmacologia , Regeneração Hepática , Fígado/fisiologia , Animais , Northern Blotting , Clonagem Molecular , Cicloeximida/farmacologia , DNA/genética , Epitélio/fisiologia , Fibroblastos/fisiologia , Biblioteca Gênica , Técnicas In Vitro , Camundongos , RNA Mensageiro/genética , Ratos , Fatores de Tempo , Transcrição Gênica
12.
J Biol Chem ; 265(35): 21914-21, 1990 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-2123877

RESUMO

Immediate-early genes, whose expression increases independent of de novo protein synthesis during the transition from quiescence to proliferation, are postulated to play important regulatory roles in the growth response. The complement of immediate-early genes expressed must depend on the milieu of preexisting transcription factors in the quiescent cell as well as the type of mitogenic stimulation and, thus, may differ between cell types. We have begun characterizing the immediate-early response in regenerating liver and insulin-stimulated Reuber H-35 hepatoma cells in comparison with previously published results from mitogen-stimulated Balb/c 3T3 fibroblasts. The proliferating H-35 and regenerating liver cells maintain their similarity to quiescent liver as demonstrated by their continued production of the liver-specific albumin, CCAAT/enhancer binding protein, and phosphoenolpyruvate carboxykinase messenger RNAs (mRNA). Surprisingly, the phosphoenolpyruvate carboxykinase gene, which undergoes down-regulation in insulin-treated H-35 cells, was cloned by differential screening of a subtraction-enriched regenerating liver cDNA library and is an immediate-early gene in regenerating liver. H-35 cells treated with either insulin or phorbol 12-myristate 13-acetate express elevated levels of the jun genes, and phorbol 12-myristate 13-acetate pretreatment fails to abolish the insulin response, indicating that it does not depend on protein kinase C. jun family gene expression in regenerating liver differs from that in mitogen-treated fibroblasts in that the time course of expression of c-jun and junB is prolonged, and junD mRNA levels distinctly increase. Additionally, although c-fos and egr-1 mRNAs are expressed at elevated levels in stimulated liver cells, fos-B, fra-1, and egr-2 are not, which suggests that factors in addition to the serum response factor participate in the regulation of immediate-early gene induction. Interestingly, gene 33, which was cloned from a regenerating liver cDNA library by differential screening and lacks a recognizable serum response element, functions as an immediate-early gene in regenerating liver and in mitogen-treated H-35 and Balb/c 3T3 cells. These results suggest that gene 33 participates in the transition from quiescence to proliferation in many mitogen-treated cells in addition to its previously reported involvement in hormone responses. Overall, the results presented here suggest that the immediate-early response varies considerably between regenerating liver and mitogen-stimulated fibroblasts and could involve multiple, preexisting, tissue-specific, transcription-activating proteins.


Assuntos
Divisão Celular , Expressão Gênica , Insulina/farmacologia , Regeneração Hepática , Fígado/fisiologia , Mitógenos/farmacologia , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , Animais , Northern Blotting , Proteínas Estimuladoras de Ligação a CCAAT , Células Cultivadas , Proteínas de Ligação a DNA/genética , Fibroblastos/fisiologia , Técnicas In Vitro , Camundongos , Família Multigênica , Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-fos , Proteínas Proto-Oncogênicas c-jun , Ratos , Albumina Sérica/genética , Acetato de Tetradecanoilforbol/farmacologia , Distribuição Tecidual , Fatores de Transcrição/genética
13.
Mol Cell Biol ; 8(10): 4533-6, 1988 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-3141793

RESUMO

The quantitative levels of initiation of protein synthesis at codons other than AUG were determined with a CYC7-lacZ fused gene in the yeast Saccharomyces cerevisiae. AUG was the only codon which efficiently initiated translation, although some non-AUG codons allowed initiation at very low efficiency, below 1% of the normal level. Since translation initiates at codons other than AUG in at least two wild-type genes from eucaryotes, other factors presumably play a role in enhancing the activity of non-AUG codons.


Assuntos
Iniciação Traducional da Cadeia Peptídica , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Sequência de Bases , Clonagem Molecular , Códon , Análise Mutacional de DNA , Dados de Sequência Molecular , RNA Fúngico/genética , Transcrição Gênica , beta-Galactosidase/genética
14.
Proc Natl Acad Sci U S A ; 81(14): 4475-9, 1984 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-6087325

RESUMO

The two unlinked genes CYC1 and CYC7 encode iso-1-cytochrome c and iso-2-cytochrome c, respectively, in the yeast Saccharomyces cerevisiae. An examination of the steady-state level of CYC1 and CYC7 mRNAs in normal and mutant strains grown under different conditions, along with previous results of apoprotein levels, demonstrate that CYC1 and CYC7 have similar and different modes of regulation. Both CYC1 and CYC7 mRNAs are diminished after anaerobic growth. In contrast, CYC1 mRNA but not CYC7 mRNA is decreased by heme deficiency in hem1 mutants. Although both CYC1 and CYC7 mRNAs are substantially lowered after growth in glucose medium, there is a difference in the kinetics of glucose derepression. CYC1 mRNA levels rise in the early logarithmic phase of growth before complete exhaustion of glucose, whereas CYC7 mRNA levels rise in the late logarithmic phase when the level of CYC1 mRNA has plateaued. For a brief period before cessation of growth, the level of CYC7 mRNA attains a level corresponding to the high derepressed level of CYC1 mRNA. The high amount of CYC7 mRNA is surprising because iso-2-cytochrome c constitutes only 5% of the total cytochrome c complement in derepressed cells. We suggest that iso-2-cytochrome c has the potential to comprise a major proportion of cytochrome c under certain physiologic conditions that have not been experimentally defined. The cyc3 mutant, which lacks the ability to attach heme groups to apocytochromes c, contains both CYC1 and CYC7 mRNAs in normal amounts. Yet, cyc3 mutants contain only apoiso-2-cytochrome c and not apoiso-1-cytochrome c. The lack of accumulation of apoiso-1-cytochrome c in cyc3 mutants, which contain CYC1 mRNA, suggests that apoiso-1-cytochrome c is extensively regulated by a post-transcriptional process.


Assuntos
Grupo dos Citocromos c/análogos & derivados , Citocromos c , Regulação da Expressão Gênica , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Grupo dos Citocromos c/genética , Glucose/farmacologia , Heme/metabolismo , Mutação , RNA Mensageiro/metabolismo
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