RESUMO
During the last 2 weeks of February 1977, an intensive scientific investigation of the martian satellite Phobos was conducted by the Viking Orbiter-1 (VO-1) spacecraft. More than 125 television pictures were obtained during this period and infrared observations were made. About 80 percent of the illuminated hemisphere was imaged at a resolution of about 30 meters. Higher resolution images of limited areas were also obtained. Flyby distances within 80 kilometers of the surface were achieved. An estimate of the mass of Phobos (GM) was obtained by observing the effect of Phobos's gravity on the orbit of VO-1 as sensed by Earth-based radiometric tracking. Preliminary results indicate a value of GM of 0.00066 +/- 0.00012 cubic kilometer per second squared (standard deviation of 3) and a mean density of about 1.9 +/- 0.6 gram per cubic centimeter (standard deviation of 3). This low density, together with the low albedo and the recently determined spectral reflectance, suggest that Phobos is compositionally similar to type I carbonaceous chondrites. Thus, either this object formed in the outer part of the asteroid belt or Lewis's theory that such material cannot condense at 1.5 astronomical units is incorrect. The data on Phobos obtained during this first encounter period are comparable in quantity to all of the data on Mars returned by Mariner flights 4, 6, and 7.
RESUMO
The structure and expression of the c-myc oncogene were examined in 29 primary human colon adenocarcinomas. Dot blot hybridization of total RNA showed that 21 tumors (72%) had considerably elevated expression of c-myc (5- to 40-fold) relative to normal colonic mucosa. These data were corroborated by Northern blots of polyadenylated RNA, which showed a 2.3-kilobase transcript. Southern analysis of the c-myc locus in these tumors indicated the absence of amplification or DNA rearrangement in a 35-kilobase region encompassing the gene. In a parallel study, elevated expression of c-myc without amplification or DNA rearrangement was also observed in three of six colon carcinoma cell lines examined; in addition, unlike a normal colon cell line control, these three cell lines exhibited constitutive, high-level expression of the gene during their growth in cultures. These results indicate that elevated expression of the c-myc oncogene occurs frequently in primary human colon carcinomas and that the mechanism involved in the regulation of c-myc expression is altered in tumor-derived cell lines.
Assuntos
Neoplasias do Colo/genética , Genes Reguladores , Oncogenes , Linhagem Celular , Células Cultivadas , Colo/metabolismo , Amplificação de Genes , Humanos , Hibridização de Ácido Nucleico , RNA Neoplásico/isolamento & purificação , Transcrição GênicaRESUMO
c-myc is the cellular gene homologous to the transforming sequence of MC29, an acute avian retrovirus. The human c-myc gene was cloned and used to study the structure and expression of c-myc in a variety of human hematopoietic malignancies. In a careful study of 106 patients, c-myc RNA was found to be expressed at elevated levels in tumor cells of 17 leukemia patients and five lymphoma patients. The c-myc gene was found to be rearranged in two lymphomas, an African Burkitt's lymphoma and a non-Hodgkins lymphoma in leukemic phase. The Burkitt's rearrangement involved the insertion of new DNA sequences upstream from the c-myc 5' coding region, presumably replacing the normal c-myc transcriptional promoter. None of the other 104 patients, including 20 with elevated myc expression, exhibited any evidence of a genetic rearrangement involving the c-myc gene. Our results show that there is a subset of hematopoietic malignancies characterized by elevated expression of c-myc. This elevated expression in most cases is not due to obvious genetic changes (rearrangement, amplification) at the c-myc locus nor to chromosomal translocations in the vicinity of this gene.
Assuntos
Clonagem Molecular , Leucemia/genética , Linfoma/genética , Oncogenes , Sequência de Bases , Linfoma de Burkitt/genética , Enzimas de Restrição do DNA , Humanos , Hibridização de Ácido Nucleico , Valores de ReferênciaRESUMO
For Ras oncoproteins to transform mammalian cells, they must be posttranslationally modified with a farnesyl group in a reaction catalyzed by the enzyme farnesyl:protein transferase (FPTase). Inhibitors of FPTase have therefore been developed as potential anticancer agents. These compounds reverse many of the malignant phenotypes of Ras-transformed cells in culture and inhibit the growth of tumor xenografts in nude mice. Furthermore, the FPTase inhibitor (FTI) L-744,832 causes tumor regression in mouse mammary tumor virus (MMTV)-v-Ha-ras transgenic mice and tumor stasis in MMTV-N-ras mice. Although these data support the further development of FTIs, it should be noted that Ki-ras is the ras gene most frequently mutated in human cancers. Moreover, Ki-RasB binds more tightly to FPTase than either Ha- or N-Ras, and thus higher concentrations of FTIs that are competitive with the protein substrate may be required to inhibit Ki-Ras processing. Given the unique biochemical and biological features of Ki-RasB, it is important to evaluate the efficacy of FTIs or any other modulator of oncogenic Ras function in model systems expressing this Ras oncoprotein. We have developed strains of transgenic mice carrying the human Ki-rasB cDNA with an activating mutation (G12V) under the control of the MMTV enhancer/promoter. The predominant pathological feature that develops in these mice is the stochastic appearance of mammary adenocarcinomas. High levels of the Ki-rasB transgene RNA are detected in these tumors. Treatment of MMTV-Ki-rasB mice with L-744,832 caused inhibition of tumor growth in the absence of systemic toxicity. Although FPTase activity was inhibited in tumors from the treated mice, unprocessed Ki-RasB was not detected. These results demonstrate the utility of the MMTV-Ki-rasB transgenic mice for testing potential anticancer agents. Additionally, the data suggest that although the FTI L-744,832 can inhibit tumor growth in this model, Ki-Ras may not be the sole mediator of the biological effects of the FTI.
Assuntos
Alquil e Aril Transferases/antagonistas & inibidores , Antineoplásicos/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Genes ras , Inibidores do Crescimento/uso terapêutico , Neoplasias Mamárias Animais/tratamento farmacológico , Vírus do Tumor Mamário do Camundongo , Metionina/análogos & derivados , Animais , Modelos Animais de Doenças , Farnesiltranstransferase , Feminino , Humanos , Metionina/uso terapêutico , Camundongos , Camundongos Transgênicos , Fenótipo , TransgenesRESUMO
[3H]Substance P was used to label putative substance P receptors in rat brain membranes. [3H]Substance P binding was saturable, reversible, and displaceable by non-radioactive substance P. Association, dissociation and saturation experiments suggest a single site interaction of [3H]substance P to its binding site (k-1/k+1 = 0.56 nM; KD = 0.9 nM, Bmax 86.5 fmol/mg protein.
Assuntos
Encéfalo/metabolismo , Receptores de Superfície Celular/metabolismo , Substância P/metabolismo , Animais , Técnicas In Vitro , Masculino , Ratos , Ratos Endogâmicos , Receptores da Neurocinina-1Assuntos
Alquil e Aril Transferases , Clonagem Molecular/métodos , Transferases/biossíntese , Sequência de Aminoácidos , Sequência de Bases , Cromatografia por Troca Iônica/métodos , Primers do DNA , Epitopos , Escherichia coli , Humanos , Indicadores e Reagentes , Cinética , Substâncias Macromoleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Plasmídeos , Reação em Cadeia da Polimerase/métodos , Biossíntese de Proteínas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Transferases/imunologia , Transferases/isolamento & purificaçãoRESUMO
The human gene for the recently identified 5-lipoxy-genase-activating protein (FLAP) has been cloned. The gene was isolated from two different genomic libraries and is contained within four overlapping bacteriophage clones. The gene spans greater than 31 kilobases and consists of five small exons and four large introns. Southern blot analysis of human genomic DNA suggests the presence of a single FLAP gene per haploid genome. A restriction site polymorphism was identified in intron II of the gene. This restriction fragment length polymorphism appears to be present in the normal population at a fairly high frequency. The transcription initiation site was located, at an adenine residue, 74 base pairs upstream of the ATG initiation codon. Examination of the sequence of the gene 5' to the mRNA start site revealed the presence of a possible TATA box (TGTAAT) 22 base pairs upstream and potential AP-2 and glucocorticoid receptor binding sites. Functional analysis of the FLAP gene promoter was assayed by transient transfection of mouse P388D1 cells (macrophage) and human HepG2 cells (hepatoma) with 5'-flanking sequences of the FLAP gene fused upstream of the chloramphenicol acetyltransferase reporter gene. Expression in the mouse macrophage cell line of the various FLAP gene promoter constructs revealed both tissue specificity and enhancer-like activities whereas in the hepatoma cell line only a minimum level of activity was obtained.
Assuntos
Proteínas de Transporte/genética , Proteínas de Membrana/genética , Regiões Promotoras Genéticas , Proteínas Ativadoras de 5-Lipoxigenase , Sequência de Aminoácidos , Animais , Sequência de Bases , Southern Blotting , Proteínas de Transporte/metabolismo , Linhagem Celular , Clonagem Molecular , DNA , Éxons , Humanos , Íntrons , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Polimorfismo de Fragmento de Restrição , Mapeamento por Restrição , TATA Box , Transcrição Gênica , Transfecção , Células Tumorais CultivadasRESUMO
The N-termini of members of the T-cell factor (Tcf) and lymphocyte-enhancement factor (Lef) protein families bind to beta-catenin, forming bipartite transcription factors which regulate expression of genes involved in organismal development and the growth of normal and malignant colon epithelium. Elevated levels of Tcf4:beta-catenin are found in colon tumor cells with mutations in the adenomatous polyposis coli (APC) gene. The elevated levels of Tcf4:beta-catenin result in increased transcription of genes, including c-myc, important for the growth of these tumor cells. Here we analyze the interaction between beta-catenin and Tcf4 and show that the N-terminal 53 amino acids of Tcf4 bind with high affinity to beta-catenin. We show that this high-affinity interaction involves multiple contact points including Tcf4 Asp-16, which is essential for beta-catenin binding. In addition to Tcf/Lef family members, beta-catenin binds to APC and cadherins. We found that the binding of beta-catenin to Tcf4, APC, or E-cadherin was mutually exclusive. These results are discussed with regard to how beta-catenin interacts with its binding partners and to the potential for identifying specific, small molecule inhibitors of these interactions.
Assuntos
Aminoácidos/metabolismo , Proteínas do Citoesqueleto/metabolismo , Transativadores , Fatores de Transcrição/metabolismo , Proteína da Polipose Adenomatosa do Colo , Sequência de Aminoácidos , Substituição de Aminoácidos , Aminoácidos/análise , Aminoácidos/genética , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Ligação Competitiva , Caderinas/química , Caderinas/genética , Caderinas/metabolismo , Sequência Conservada , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Ensaio de Imunoadsorção Enzimática , Humanos , Concentração Inibidora 50 , Leucina/genética , Leucina/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , Solubilidade , Fatores de Transcrição TCF , Proteína 2 Semelhante ao Fator 7 de Transcrição , Fatores de Transcrição/química , Fatores de Transcrição/genética , beta CateninaRESUMO
The enzyme 5-lipoxygenase (5-LO) catalyzes the first two reactions in the pathway leading to the formation of leukotrienes from arachidonic acid. Leukotrienes are potent arachidonic acid metabolites possessing biological activities that suggest a role in the pathophysiology of allergic and inflammatory diseases. To obtain structural information about 5-LO for use in developing anti-inflammatory chemotherapeutic agents, the enzyme was purified from human polymorphonuclear leukocytes and the amino acid sequences were determined for several cyanogen bromide-derived peptides. A cDNA clone encoding a 674-amino acid protein containing all of the derived peptide sequences was isolated from a dimethyl sulfoxide differentiated HL60 cell cDNA library. The mRNA encoding 5-LO was detected only in differentiated HL60 cells and not in the undifferentiated cell line, indicating that the expression of 5-LO in this cell line is transcriptionally regulated. In addition, the human protein displays some amino acid sequence homology with several lipases and significant homology with the partial sequences of soybean and reticulocyte lipoxygenases. Thus, 5-LO appears to be a member of a larger family of related enzymes.
Assuntos
Araquidonato 5-Lipoxigenase/genética , Araquidonato Lipoxigenases/genética , Clonagem Molecular , Genes , Sequência de Aminoácidos , Animais , Araquidonato 5-Lipoxigenase/sangue , Araquidonato 5-Lipoxigenase/isolamento & purificação , Sequência de Bases , Linhagem Celular , DNA/sangue , DNA/genética , Humanos , Dados de Sequência Molecular , Neutrófilos/enzimologia , Homologia de Sequência do Ácido NucleicoRESUMO
A series of compounds that inhibit the coupling of the alpha 2-adrenergic receptor and the beta 2-adrenergic receptor to the guanine nucleotide-binding proteins (G proteins) Gi and Gs, respectively, have been identified. This inhibition of G protein coupling was detected by the ability of the compounds to reduce the affinity of these receptors for agonists without affecting antagonist affinity. Analysis of the structure-activity relationships of these compounds revealed a requirement for regularly spaced anionic substituents on amphipathic structures for this inhibition to occur. The compounds do not interact at the ligand binding site of the receptor or at the GTP binding site of the G protein. The identification of compounds that can uncouple receptors from G proteins demonstrates the potential for the discovery of small molecule inhibitors of receptor-G protein interactions that act as allosteric antagonists at this site.
Assuntos
Proteínas de Ligação ao GTP/efeitos dos fármacos , Receptores Adrenérgicos alfa/efeitos dos fármacos , Receptores Adrenérgicos beta/efeitos dos fármacos , Regulação Alostérica/efeitos dos fármacos , Animais , Linhagem Celular , Proteínas de Ligação ao GTP/metabolismo , Humanos , Naftalenossulfonatos/farmacologia , Ligação Proteica/efeitos dos fármacos , Receptores Adrenérgicos alfa/metabolismo , Receptores Adrenérgicos beta/metabolismo , Relação Estrutura-Atividade , Simpatolíticos/metabolismo , Simpatomiméticos/metabolismoRESUMO
The roles of 11 conserved amino acids of the beta-subunit of human farnesyl:protein transferase (FTase) were examined by performing kinetic and biochemical analyses of site-directed mutants. This biochemical information along with the x-ray crystal structure of rat FTase indicates that residues His-248, Arg-291, Lys-294, and Trp-303 are involved with binding and utilization of the substrate farnesyl diphosphate. Our data confirm structural evidence that amino acids Cys-299, Asp-297, and His-362 are ligands for the essential Zn2+ ion and suggest that Asp-359 may also play a role in Zn2+ binding. Additionally, we demonstrate that Arg-202 is important for binding the essential C-terminal carboxylate of the protein substrate.
Assuntos
Alquil e Aril Transferases/química , Alquil e Aril Transferases/metabolismo , Alquil e Aril Transferases/biossíntese , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Arginina , Sequência de Bases , Sequência Conservada , Cristalografia por Raios X , Farnesiltranstransferase , Histidina , Humanos , Cinética , Lisina , Substâncias Macromoleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , TriptofanoRESUMO
The protease of human immunodeficiency virus has been expressed in Escherichia coli and purified to apparent homogeneity. Immunoreactivity toward anti-protease peptide sera copurified with an activity that cleaved the structural polyprotein gag p55 and the peptide corresponding to the sequence gag 128-135. The enzyme expressed as a nonfusion protein exhibits proteolytic activity with a pH optimum of 5.5 and is inhibited by the aspartic protease inhibitor pepstatin with a Ki of 1.1 microM. Replacement of the conserved residue Asp-25 with an Asn residue eliminates proteolytic activity. Analysis of the minimal peptide substrate size indicates that 7 amino acids are required for efficient peptide cleavage. Size exclusion chromatography is consistent with a dimeric enzyme and circular dichroism spectra of the purified enzyme are consistent with a proposed structure of the protease (Pearl, L.H., and Taylor, W.R. (1987) Nature 329, 351-354). These data support the classification of the human immunodeficiency virus protease as an aspartic protease, likely to be structurally homologous with the well characterized family that includes pepsin and renin.
Assuntos
Endopeptidases/genética , HIV-1/genética , Ácido Aspártico Endopeptidases , Cromatografia por Troca Iônica , Clonagem Molecular , DNA Recombinante/metabolismo , Endopeptidases/isolamento & purificação , Endopeptidases/metabolismo , Escherichia coli/genética , Genes , Genes Virais , HIV-1/enzimologia , Immunoblotting , Cinética , Peso Molecular , Plasmídeos , Especificidade por SubstratoRESUMO
Pharmacological analysis of ligand binding to the beta-adrenergic receptor (beta AR) has revealed the existence of two distinct receptor subtypes (beta 1 and beta 2) which are the products of different genes. The predicted amino acid sequences of the beta 1 and beta 2 receptors differ by 48%. To identify the regions of the proteins responsible for determining receptor subtype, chimeras were constructed from domains of the human beta 1 and hamster beta 2 receptors. Analysis of the ligand-binding characteristics of these hybrid receptors revealed that residues in the middle portion of the beta AR sequence, particularly around transmembrane regions 4 and 5, contribute to the subtype specific binding of agonists. Smaller molecular replacements of regions of the hamster beta 2 AR with the analogous regions from the avian beta 1 AR, however, failed to identify any single residue substitution capable of altering the subtype specificity of the receptor. These data indicate that, whereas sequences around transmembrane regions 4 and 5 may contribute to conformations which influence the ligand-binding properties of the receptor, the subtype-specific differences in amine-substituted agonist binding cannot be attributed to a single molecular interaction between the ligand and any amino acid residue which is divergent between the beta 1 and beta 2 receptors.
Assuntos
Receptores Adrenérgicos beta/genética , Sequência de Aminoácidos , Animais , Células Cultivadas , Cricetinae , DNA/biossíntese , Humanos , Dados de Sequência Molecular , Fenótipo , Proteínas Recombinantes , PerusRESUMO
In conclusion, we have cloned a full-length cDNA for human leukocyte 5-LO from differentiating HL-60 cells. The complete amino acid sequence of 5-LO has been determined from the nucleotide sequence of the cDNA. Some interesting features of the sequence include potential lipid and Ca2+ binding sites and sequence homologies with other lipoxygenases. Human osteosarcoma cells transfected with the 5-LO cDNA expressed 5-LO and LTA4 synthase activities that were indistinguishable from those of the human leukocyte enzyme confirming that the cloned cDNA was the correct gene.
Assuntos
Araquidonato 5-Lipoxigenase/genética , Araquidonato Lipoxigenases/genética , Clonagem Molecular , Genes , Leucócitos/enzimologia , Sequência de Aminoácidos , Araquidonato 5-Lipoxigenase/biossíntese , Linhagem Celular , Humanos , Dados de Sequência Molecular , TransfecçãoRESUMO
Aspartyl (asparaginyl) beta-hydroxylase which specifically hydroxylates 1 Asp or Asn residue in certain epidermal growth factor-like domains of a number of proteins, has been previously purified to apparent homogeneity from detergent-solubilized bovine liver microsomes (Wang, Q., VanDusen, W. J., Petroski, C. J., Garsky, V. M., Stern, A. M., and Friedman, P. A. (1991) J. Biol. Chem. 266, 14004-14010). Three oligonucleotides, corresponding to three amino acid sequences of the purified hydroxylase, were used to screen bovine cDNA libraries. Several overlapping positive cDNA clones containing a full length open reading frame of 754 amino acids encoding a 85-kDa protein were isolated, and a cDNA, containing the full length open reading frame, was constructed from two of these clones. The resulting clone was then transcribed and translated in vitro to produce recombinant protein which possessed Asp beta-hydroxylase activity. These results constitute proof that the protein purified from bovine liver is an Asp beta-hydroxylase. Comparisons of deduced amino acid sequences of two other alpha-ketoglutarate-dependent dioxygenases, prolyl-4-hydroxylase and lysyl hydroxylase, with that of Asp beta-hydroxylase showed no significant homologies. Indeed, Asp beta-hydroxylase appears to be unique as no striking homology was found with known protein sequences. Furthermore, structural predictions derived from the deduced amino acid sequence are in accord with earlier Stokes' radius and sedimentation coefficient determinations of the enzyme, suggesting that the enzyme contains a relatively compact carboxyl-terminal catalytic domain and an extended amino terminus. This amino-terminal region has a potential transmembrane type II signal-anchor domain that could direct the catalytic domain into the lumen of the endoplasmic reticulum.
Assuntos
DNA/genética , Fígado/enzimologia , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Bovinos , Deleção Cromossômica , Clonagem Molecular , DNA/isolamento & purificação , Cinética , Oxigenases de Função Mista/isolamento & purificação , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Plasmídeos , Poli A/genética , Poli A/isolamento & purificação , Biossíntese de Proteínas , RNA/genética , RNA/isolamento & purificação , RNA Mensageiro , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Homologia de Sequência do Ácido NucleicoRESUMO
We have isolated cDNAs encoding the alpha and beta subunits of human farnesyl-protein transferase (FPTase). The proteins encoded by these two cDNAs are 93-95% identical to the corresponding subunits of bovine and rat FPTase and show regions of homology with proteins encoded by Saccharomyces cerevisiae prenyl-protein transferase genes. Human FPTase expressed in Escherichia coli from a translationally coupled operon had kinetic properties similar to those of FPTase isolated from bovine brain. Examination of farnesyl diphosphate binding indicated that while neither individual subunit was capable of isoprenoid binding, a radiolabeled farnesyl diphosphate analog could be specifically photo-cross-linked to the beta subunit of FPTase holoenzyme. To further analyze subunit structure-function and to detect functional similarities with yeast prenyl-protein transferases (FPTase and two geranylgeranyl-protein transferases), amino acid changes homologous to those found in mutant yeast prenyl-protein transferase subunits were made in the subunits of human FPTase. Substitutions in either the alpha or beta subunits that decrease the activity of yeast prenyl-protein transferases were also observed to impair human FPTase. Kinetic analyses showed that these mutant human FPTases have Km and kcat values that are altered with respect to wild-type human FPTase.
Assuntos
Alquil e Aril Transferases , Clonagem Molecular , Expressão Gênica , Fosfatos de Poli-Isoprenil/metabolismo , Saccharomyces cerevisiae/enzimologia , Transferases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , DNA/química , Escherichia coli/genética , Farnesiltranstransferase , Humanos , Cinética , Dados de Sequência Molecular , Mutação , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Sesquiterpenos , Relação Estrutura-Atividade , Transferases/química , Transferases/metabolismoRESUMO
The plasma membrane-bound mammalian ras proteins of relative molecular mass 21,000 (ras p21) share biochemical and structural properties with other guanine nucleotide-binding regulatory proteins (G-proteins). Oncogenic ras p21 variants result from amino acid substitutions at specific positions that cause p21 to occur predominantly complexed to GTP in vivo. Recently, a GTPase activating protein (GAP) with cytosolic activity has been discovered that stimulates the GTPase activity of normal but not of oncogenic ras p21. GAP might be either a negative regulatory agent which acts further upstream in the regulatory pathway or the downstream target of ras p21. We have identified a protein from bovine brain with apparent relative molecular mass 125,000 that has GAP activity. Here, using pure GAP in a kinetic competition assay, we show that GAP interacts preferentially with the active GTP complexes of both normal and oncogenic Harvey (Ha) ras p21 compared with the inactive GDP complexes. We also report the cloning and sequencing of the complementary DNA for bovine GAP. Regions of GAP share amino acid similarity with the noncatalytic domain of adenylate cyclase from the yeast Saccharomyces cerevisiae and with regions conserved between phospholipase C-148, the crk oncogene product and the nonreceptor tyrosine kinases.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas , Proteínas Proto-Oncogênicas , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/metabolismo , Bovinos , Clonagem Molecular , DNA , Proteínas Ativadoras de GTPase , Dados de Sequência Molecular , Peso Molecular , Proteínas Ativadoras de ras GTPaseRESUMO
Protein geranylgeranyltransferase type-I (GGTase-I) transfers a geranylgeranyl group to the cysteine residue of candidate proteins containing a carboxyl-terminal CAAX (C, cysteine; A, aliphatic amino acid; X, any amino acid) motif in which the "X" residue is leucine. The enzyme is composed of a 48-kilodalton alpha subunit and a 43-kilodalton beta subunit. Peptides isolated from the alpha subunit of GGTase-I were shown to be identical with the alpha subunit of a related enzyme, protein farnesyltransferase. Overlapping cDNA clones containing the complete coding sequence for the beta subunit of GGTase-I were obtained from rat and human cDNA libraries. The cDNA clones from both species each predicted a protein of 377 amino acids with molecular masses of 42.4 kilodaltons (human) and 42.5 kilodaltons (rat). Amino acid sequence comparison suggests that the protein encoded by the Saccharomyces cerevisiae gene CDC43 is the yeast counterpart of the mammalian GGTase-I beta subunit. Co-expression of the GGTase-I beta subunit cDNA together with the alpha subunit of protein farnesyltransferase in Escherichia coli produced recombinant GGTase-I with electrophoretic and enzymatic properties indistinguishable from native GGTase-I.
Assuntos
Alquil e Aril Transferases , Proteínas de Ciclo Celular , Isoenzimas/biossíntese , Proteínas de Saccharomyces cerevisiae , Transferases/biossíntese , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular/métodos , DNA Complementar/metabolismo , Dimetilaliltranstransferase/genética , Farnesiltranstransferase , Proteínas Fúngicas/genética , Expressão Gênica , Biblioteca Gênica , Genes Fúngicos , Humanos , Isoenzimas/genética , Isoenzimas/isolamento & purificação , Substâncias Macromoleculares , Dados de Sequência Molecular , Ratos , Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Transferases/genética , Transferases/isolamento & purificaçãoRESUMO
Inositol monophosphatase is a key enzyme of the inositol phosphate second messenger signaling pathway. It is responsible for the provision of inositol required for synthesis of phosphatidylinositol and polyphosphoinositides and has been implicated as the pharmacological target for lithium action in brain. Using oligonucleotide probes based on partial amino acid sequence data for the bovine brain enzyme, several overlapping cDNA clones of 2-3 kilobases in length have been isolated. All contain an open reading frame encoding a 277-amino acid protein. No significant sequence homology was found with any known protein. The open reading frame was inserted into a bacterial expression vector in order to confirm the presumed identity of the protein. The expressed protein reacted with an anti-inositol monophosphatase monoclonal antibody. In addition, the protein was enzymically active and indistinguishable from the bovine brain enzyme with respect to Km values for substrate and Li+ sensitivity of inositol 1-phosphate hydrolysis.
Assuntos
Córtex Cerebral/enzimologia , Genes , Monoéster Fosfórico Hidrolases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Clonagem Molecular , DNA/genética , Escherichia coli/genética , Expressão Gênica , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Monoéster Fosfórico Hidrolases/isolamento & purificação , Monoéster Fosfórico Hidrolases/metabolismo , RNA/genética , RNA/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Mapeamento por RestriçãoRESUMO
To generate nonpathogenic viral particles which express active human immunodeficiency virus type 1 (HIV-1) protease (PR), plasmids containing sequences from the genomes of HIV-1 and Moloney murine leukemia virus (M-MuLV) were constructed. Either the PR coding region alone; the gag, PR, and reverse transcriptase protein-coding regions; or the complete gag and pol protein-coding regions from HIV-1 were substituted for the corresponding regions of a full-length M-MuLV clone to yield the chimeric plasmids pMoHIV-I, pMoHIV-III, and pMoHIV-IV, respectively. Cell lines which express the viral gag polyprotein were isolated for hybrids pMoHIV-I and pMoHIV-III. These cells produced viral particles which contained processed core proteins. Cleavage of the gag polyprotein in the viral particles was inhibited by the HIV-1 PR inhibitor L-687908, indicating that the viral PR is responsible for the observed processing. The hybrid virions were not infectious; analyses indicated that the viral particles contained little or no reverse transcriptase activity. In addition, particles produced by pMoHIV-III transfectants failed to package the viral genomic RNA. The cell line which expresses and processes the HIV-1 gag polyprotein is a safe and effective reagent for the in vivo evaluation of potential inhibitors of the HIV-1 PR.