RESUMO
Human apolipoprotein A-IV (apo A-IV) is essentially synthesized in intestine and to a lower extent in liver. The presence of detectable amounts of apo A-IV messenger in HepG2 and Caco-2 cells enables us to study the transcription regulation of the gene in these cells. A main hypersensitive site to DNase I is detected in the DNA of HepG2 nuclei, in a region situated about 3400 base pairs upstream from the cap site. Transient expression experiments in HepG2, Caco-2 and HeLa cells indicate that HNF-4 activates the transcription of a reporter gene through the -3500/+67 sequence of the apo A-IV gene. This activation is repressed by another transcription factor, Ear3/COUP-TF. On the contrary, HNF-1, whose expression is controlled by HNF-4, is not involved in the regulation of the reporter gene transcription through the -6700/+67 genome sequence. These results indicate that the apo A-IV gene expression is modulated by orphan ligand members of the superfamily of nuclear hormone receptors.
Assuntos
Apolipoproteínas A/genética , Regulação da Expressão Gênica , Fosfoproteínas , Fatores de Transcrição/metabolismo , Apolipoproteínas A/biossíntese , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Fator I de Transcrição COUP , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Desoxirribonuclease I , Vetores Genéticos , Células HeLa , Fator 4 Nuclear de Hepatócito , Humanos , Especificidade de Órgãos/genética , Transcrição Gênica , TransfecçãoRESUMO
The human iron-binding protein melanotransferrin is up-regulated in most skin melanomas. With the goal to understand the mechanisms controlling the expression of the gene in these tumor cells, we previously reported the identification of an enhancer exhibiting melanoma specificity. We show here that, in the highly expressing SK-MEL-28 melanoma cell line, the chromatin structure in the enhancer region is in an open configuration and that the transcription factors governing its activity belong to the helix-loop-helix and the Jun/Fos leucine zipper families.
Assuntos
Antígenos de Neoplasias/genética , Elementos Facilitadores Genéticos , Regulação Neoplásica da Expressão Gênica , Melanoma/genética , Proteínas de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Sequência de Bases , Sítios de Ligação , DNA de Neoplasias , Desoxirribonuclease I/metabolismo , Células HeLa , Sequências Hélice-Alça-Hélice , Humanos , Antígenos Específicos de Melanoma , Dados de Sequência Molecular , Proteínas de Neoplasias/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Células Tumorais CultivadasRESUMO
Genetic, epidemiological and clinical evidence have clearly demonstrated the importance of the human apolipoprotein (apo) A-I/C-III/A-IV gene cluster in lipid metabolism and heart attack. The transcriptional regulation of these genes determines the level of the encoded proteins and therefore influences the concentration of triglycerides and cholesterol. Here, we analyze the existence of transcription control elements in the 6.6 kb apoC-III/A-IV intergenic region and their influence on the expression of both genes. Two main positive common control elements were found to modulate apoC-III and apoA-IV expression in HepG2 and in Caco-2 cells: the previously described apoC-III enhancer, located 0.8 kb upstream from the cap site of the gene, and a newly detected activating region located in the center of the intergenic sequence. The activity of both elements is highly increased by the hepatic and intestinal transcription factor HNF-4. Analysis of a 641 bp fragment containing the central element showed that it has the properties of a tissue-specific enhancer. Liver nuclear proteins interact with seven DNA binding sites present in this enhancer and HNF-4 specifically interacts with one of these sites. A third positive element, situated immediately upstream from the apoA-IV minimal promoter, is also activated by HNF-4; however, this element is not involved in apoC-III expression. In addition, two negative regions were identified, one located near the apoA-IV gene and the other one between the apoC-III enhancer and the newly identified central enhancer. In conclusion, negative and positive control elements are located in the apoC-III/A-IV intergenic region, including two enhancers important for the expression of the two genes. These results add new evidence that common regulatory elements for the expression of the apoA-I, apoC-III and apoA-IV genes are interspersed throughout the cluster.
Assuntos
Apolipoproteína A-I/genética , Apolipoproteínas A/genética , Apolipoproteínas C/genética , Proteínas de Ligação a DNA , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Família Multigênica , Apolipoproteína C-III , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Carcinoma Hepatocelular/metabolismo , Neoplasias do Colo/metabolismo , Desoxirribonuclease I , Expressão Gênica , Vetores Genéticos , Fator 4 Nuclear de Hepatócito , Humanos , Neoplasias Hepáticas/metabolismo , Dados de Sequência Molecular , Fosfoproteínas/genética , Plasmídeos , Fatores de Transcrição/genética , Transfecção , Células Tumorais CultivadasRESUMO
Mice expressing human apolipoprotein A-IV (apoA-IV) mainly in the intestine were obtained in an apolipoprotein E-deficient (apoE(0)) background (apoA-IV/E(0) mice). Quantification of aortic lesions and plasma lipid determination showed that compared with their control apoE(0) counterparts, the apoA-IV/E(0) mice are protected against atherosclerosis without an increase in HDL cholesterol. Because oxidized lipoproteins play an important role in atherogenesis, we tested whether the protection observed in these animals is accompanied by an in vivo reduction of the oxidation parameters. The lag time in the formation of conjugated dienes during copper-mediated oxidation, the aggregation state of LDL, and the presence of anti-oxidized LDL antibodies were measured. The presence of oxidized proteins in tissues and the presence of oxidation-specific epitopes in heart sections of atherosclerotic lesions were also analyzed. Except for lag time, the results showed that the oxidation parameters were reduced in the apoA-IV/E(0) mice compared with the apoE(0) mice. This suggests that human apoA-IV acts in vivo as an antioxidant. In addition, human apoA-IV accumulation was detected in the atherosclerotic lesions of apoA-IV/E(0) mice, suggesting that apoA-IV may inhibit oxidative damage to local tissues, thus decreasing the progression of atherosclerosis.
Assuntos
Antioxidantes , Apolipoproteínas A/genética , Apolipoproteínas E/genética , Arteriosclerose/prevenção & controle , Animais , Anticorpos Monoclonais/imunologia , Apolipoproteínas A/metabolismo , Arteriosclerose/metabolismo , Arteriosclerose/patologia , HDL-Colesterol/sangue , Feminino , Humanos , Mucosa Intestinal/metabolismo , Lipoproteínas/imunologia , Lipoproteínas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , OxirreduçãoRESUMO
The apolipoprotein (apo)A-I/C-III/A-IV gene cluster is involved in lipid metabolism and atherosclerosis. Overexpression of apoC-III in mice causes hypertriglyceridemia and induces atherogenesis, whereas overexpression of apoA-I or apoA-IV increases cholesterol in plasma high density lipoprotein (HDL) and protects against atherosclerosis. Each gene has been studied alone in transgenic mice but not in combination as the entire cluster. To determine which phenotype is produced by the expression of the entire gene cluster, transgenic mice were generated with a 33-kb human DNA fragment. The results showed that the transgene contained the necessary elements to direct hepatic and intestinal expression of the 3 genes. In the pooled data, plasma concentrations were 257+/-9, 7.1+/-0.5, and 1.0+/-0.2 mg/dL for human apoA-I, apoC-III, and apoA-IV, respectively (mean+/-SEM). Concentrations of these apolipoproteins were higher in males than in females. Human apoA-I and apoC-III concentrations were positively correlated, suggesting that they are coregulated. Transgenic mice exhibited gross hypertriglyceridemia and accumulation of apoB(48)-containing triglyceride-rich lipoproteins. Plasma triglyceride and cholesterol concentrations were correlated positively with human apoC-III concentration, and HDL cholesterol was correlated with apoA-I concentration. In an apoE-deficient background, despite being markedly hypertriglyceridemic, cluster transgenic animals compared with nontransgenic animals showed a 61% reduction in atherosclerosis. This suggests that apoA-I and/or apoA-IV can protect against atherosclerosis even in the presence of severe hyperlipidemia. These mice provide a new model for studies of the regulation of the 3 human genes in combination.
Assuntos
Apolipoproteína A-I/genética , Apolipoproteínas A/genética , Apolipoproteínas C/genética , Arteriosclerose/prevenção & controle , Hiperlipidemias/genética , Animais , Apolipoproteína A-I/análise , Apolipoproteína C-III , Apolipoproteínas B/sangue , Apolipoproteínas B/química , Apolipoproteínas C/análise , Arteriosclerose/genética , HDL-Colesterol/sangue , Feminino , Regulação da Expressão Gênica , Hiperlipidemias/sangue , Lipoproteínas/sangue , Lipoproteínas/química , Masculino , Camundongos , Camundongos Transgênicos , Modelos Animais , Família Multigênica , Fatores Sexuais , Triglicerídeos/sangueRESUMO
Studies in Nur77-deficient mice have shown that the basal regulation of hypothalamic and pituitary functions as well as the adrenocortical steroidogenesis in these animals is normal. This indicates that Nur77-related orphan receptors may substitute Nur77 functions in the hypothalamo-pituitary-adrenal axis by a compensatory mechanism. Nor1 is the most recently cloned member of the NGFI-B/Nur77 subfamily, and its properties are still largely unknown. We demonstrate here that Nor1 is expressed in the pituitary gland and adrenal cortex, and that ACTH and angiotensin II (AngII) treatment of adrenal fasciculata cells induces Nor1 expression. Time-course analysis with both hormones on steroidogenic capacity and the specific gene expression in adrenal cells strongly suggest that Nor1 is an intermediate in the long-term consequences of ACTH or AngII treatment. The Nor1 and NGFI-B/Nur77 amino acid sequence homology and the analysis of the trans-activation properties of Nor1 show that the overall structural and functional organization of the two proteins is similar. As observed with NGFI-B/Nur77, Nor1 activates the expression of genes encoding steroidogenic enzymes as P450c21, through its interaction with NGFI-B response element promoter sequences. In contrast, binding experiments of Nor1 with the palindromic NurRE sequence suggest that Nor1 is not an efficient substitute for the NGFI-B/Nur77 activation of the POMC gene expression in pituitary glands. All these results indicate that Nor1 and NGFI-B/Nur77 may play similar albeit distinct roles in the hypothalamo-pituitary-adrenal axis. Further experiments also show that the mechanisms responsible for the transcriptional regulation of Nor1 in adrenal cells appear to depend on the protein kinase A and protein kinase C cascades.
Assuntos
Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/fisiologia , Sistema Hipotálamo-Hipofisário/fisiologia , Proteínas do Tecido Nervoso , Proteínas Nucleares/fisiologia , Sistema Hipófise-Suprarrenal/fisiologia , Fatores de Transcrição/fisiologia , Córtex Suprarrenal/metabolismo , Glândulas Suprarrenais/citologia , Glândulas Suprarrenais/metabolismo , Hormônio Adrenocorticotrópico/farmacologia , Angiotensina II/farmacologia , Animais , Células COS , Sistema Enzimático do Citocromo P-450/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica/fisiologia , Genes Reporter/fisiologia , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares , Hipófise/metabolismo , Pró-Opiomelanocortina/genética , Regiões Promotoras Genéticas/fisiologia , RNA Mensageiro/metabolismo , Receptores Citoplasmáticos e Nucleares , Receptores de Esteroides , Receptores dos Hormônios Tireóideos , Sistemas do Segundo Mensageiro/fisiologia , Esteroide 21-Hidroxilase , Ativação Transcricional/fisiologiaRESUMO
The genes coding for human apolipoproteins AI, CIII and AIV are tandemly organised in a cluster on chromosome 11. The sequence of 4 kb of the 6.6-kb ApoCIII-ApoAIV intergenic region was unknown until now. Since different elements involved in the transcriptional regulation of the three genes of the cluster were previously identified in this region, we decided to sequence it. We present here the complete sequence of the region. Its availability will allow study of the transcriptional regulation of ApoCIII and ApoAIV at the molecular level. In addition, the use of PCR to study the polymorphic sites detected in the ApoCIII-ApoAIV intergenic region will now be possible. Some of these sites have been associated with lipid disorders and coronary heart disease.
Assuntos
Apolipoproteínas A/genética , Apolipoproteínas C/genética , Família Multigênica/genética , Sequências Reguladoras de Ácido Nucleico/genética , Apolipoproteína C-III , Sequência de Bases , Humanos , Dados de Sequência Molecular , Sequências Repetitivas de Ácido Nucleico/genética , Análise de Sequência de DNARESUMO
The complete structure of the human transferrin gene is presented. This gene has a total size of about 33.5 kb and is organized in 17 exons separated by 16 introns. The chicken ovotransferrin gene has a size of 10.5 kb and is also organized in 17 exons and 16 introns. The analysis of the structure of the two genes confirm, at the gene level, that transferrins originated by a gene duplication phenomenon. Finally, the existence of a new member of the transferrin family, a human transferrin non-processed pseudogene is demonstrated.
Assuntos
Genes , Transferrina/genética , Sequência de Aminoácidos , Animais , Galinhas , Conalbumina/genética , DNA/genética , Éxons , Humanos , Íntrons , Dados de Sequência Molecular , Pseudogenes , Especificidade da EspécieRESUMO
The human transferrin gene enhancer is organized in two domains. Domain A contains a single enhanson designated Ia. Domain B contains four enhansons named Ib, II, III and IV. We demonstrate here that the liver-enriched transcription factor HNF-3 alpha interacts with enhanson Ia and that enhansons Ib and IV are binding sites for members of the NF1 family. In addition, enhansons II and III seem to be respectively the targets for the AP4 protein and for EIII, a factor not yet completely identified. Analysis of mutated enhancer regions establishes that each enhanson is required for full enhancer activity and that the proteins binding to enhansons II, III and IV may interact within a multiprotein complex. This enhancer region presents no activity in the Sertoli cells of testis, where transferrin is also synthesized. We demonstrate that in Sertoli cells, the members of the HNF-3 family are not expressed; this fact may account for the inactivity of the enhancer in these cells.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Transferrina/genética , Sequência de Bases , Linhagem Celular , DNA , Células HeLa , Fator 3-alfa Nuclear de Hepatócito , Humanos , Masculino , Dados de Sequência Molecular , Mutação , Ligação Proteica , Células de SertoliRESUMO
Zinc-finger proteins are involved in several cellular processes. Some of these proteins are implicated in the primary cellular response in regenerating liver and mitogen-stimulated cells. Using a rat cDNA brain library, we have isolated a clone designated NOR-2, encoding a protein containing two zinc-finger motifs and whose expression is highly induced during G0/G1 transition. We analysed the expression of NOR-2 mRNAs during early growth in regenerating liver and in both insulin-stimulated H4-II cells and pheochromocytoma-derived cell line PC12 treated by NGF. In these systems, there is an early, rapid and transient accumulation of NOR-2 mRNAs. The induction of NOR-2 mRNAs does not require de novo protein synthesis, since it is not prevented by cycloheximide treatment. Mobility shift assays show that NOR-2 protein binds to NBRE, a target sequence for r-NGFI-B family. Structurally, NOR-2 is closely related to the recently identified NOR-1 factor. Therefore, like NOR-1, NOR-2 belongs to the r-NGFI-B sub-family of nuclear receptors superfamily.
Assuntos
Encéfalo/metabolismo , Proteínas de Ligação a DNA/isolamento & purificação , Fígado/fisiologia , Proteínas do Tecido Nervoso/isolamento & purificação , Receptores de Superfície Celular/isolamento & purificação , Dedos de Zinco , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Proteínas de Ligação a DNA/genética , Masculino , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Ratos , Ratos Wistar , Receptores de Superfície Celular/genética , RegeneraçãoRESUMO
Transferrin hepato-specific gene enhancer, associated with the liver-enriched HNF-3 alpha transcriptional factor and ubiquitous proteins, is a complex molecular edifice maintained through DNA-protein and protein-protein interactions. As a first step to understand the mechanisms responsible for its organization and activity, we have analyzed the interaction of the DNA binding domain of HNF-3 alpha (HDBD) with a specific DNA segment present in the transferrin enhancer by different biophysical techniques. The kinetic constants of this interaction were measured using surface plasmon resonance. The HDBD-DNA interaction was also characterized by circular dichroism and fluorescence spectroscopy. HDBD binds to its specific DNA site with high affinity (Kd approximately equal to 10(-8) M). The affinity is reduced after sequence modification of the target DNA. Size exclusion chromatography and binding stoichiometry determined by fluorescence measurements indicate that the protein is present in a monomeric form before and after interaction with the DNA. The secondary structure of the protein was not significantly altered upon binding to specific DNA. By contrast, a structural change of DNA by interaction with HDBD seems to occur.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Elementos Facilitadores Genéticos/genética , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Transferrina/genética , Sequência de Bases , Proteínas de Ligação a DNA/química , Fator 3-alfa Nuclear de Hepatócito , Cinética , Dados de Sequência Molecular , Proteínas Nucleares/química , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
The apolipoprotein (Apo) AI-CIII-AIV gene cluster has a complex pattern of gene expression that is modulated by both gene- and cluster-specific cis-acting elements. In particular the regulation of Apo AIV expression has been previously studied in vivo and in vitro including several transgenic mouse lines but a complete, consistent picture of the tissue-specific controls is still missing. We have analysed the role of the Apo AIV 3' flanking sequences in the regulation of gene expression using both in vitro and in vivo systems including three lines of transgenic mice. The transgene consisted of a human fragment containing 7 kb of the 5' flanking region, the Apo AIV gene itself and 6 kb of the 3' flanking region (-7+6 Apo AIV). Accurate analysis of the Apo AIV mRNA levels using quantitative PCR and Northern blots showed that the 7+6 kb Apo AIV fragment confers liver-specific regulation in that the human Apo AIV transgene is expressed at approximately the same level as the endogenous mouse Apo AIV gene. In contrast, the intestinal regulation of the transgene did not follow, the pattern observed with the endogenous gene although it produced a much higher intestinal expression following the accepted human pattern. Therefore, this animal model provides an excellent substrate to design therapeutic protocols for those metabolic derangements that may benefit from variations in Apo AIV levels and its anti-atherogenic effect.
Assuntos
Apolipoproteínas A/genética , Regulação Enzimológica da Expressão Gênica , Animais , Células CACO-2 , Humanos , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase , Distribuição Tecidual , Células Tumorais CultivadasRESUMO
We have previously generated transgenic (Tg) mice expressing the human apolipoprotein (apo) A-I/C-III/A-IV gene cluster. This expression induced hyperlipidemia but reduced atherosclerotic lesions in genetically modified mice lacking apoE. Atherosclerosis is a multifactorial process and environmental factors such as diet play significant roles in its development. We examined here how an atherogenic diet influences the expression of the human genes and the characteristics of the Tg mice. Our results indicate that a high fat-high cholesterol diet up-regulates the intestinal expression of the three genes and the concentration of the three proteins in plasma. Cholesterol concentration was highly increased in the non-high density lipoprotein (HDL) fraction, and less, although significantly, in the HDL fraction. Tgs showed a 65% reduction in diet-induced aortic lesions compared with non-Tg mice. Atherogenic diet increases the expression of the genes encoding the scavenger receptor class B type I (SR-BI) and ATP binding cassette transporter 1 (ABCA1) proteins. As cholesterol efflux mediated by SR-BI or by ABCA1 was enhanced in Tg mice fed an atherogenic diet, we can hypothesize that increased reverse cholesterol transport is the basis of the protective mechanism observed in these animals. In conclusion, we present evidence that the expression of the human gene cluster in mice protects against atherogenesis in response to an atherogenic diet.
Assuntos
Apolipoproteína A-I/genética , Apolipoproteínas A/genética , Apolipoproteínas C/genética , Arteriosclerose/fisiopatologia , Expressão Gênica , Proteínas de Membrana , Família Multigênica , Receptores Imunológicos , Receptores de Lipoproteínas , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Aorta/patologia , Apolipoproteína A-I/sangue , Apolipoproteína C-III , Apolipoproteínas A/sangue , Apolipoproteínas C/sangue , Antígenos CD36/genética , Colesterol/sangue , Colesterol na Dieta/metabolismo , Gorduras na Dieta/metabolismo , Feminino , Humanos , Camundongos , Camundongos Transgênicos , Receptores Depuradores , Receptores Depuradores Classe B , Transgenes , Triglicerídeos/sangue , Células Tumorais CultivadasRESUMO
We have investigated the involvement of human apolipoprotein A-IV (apoA-IV) in gastric acid secretion and ulcer formation in recently generated apoA-IV transgenic mice. Compared to control littermates, transgenic animals showed a gastric acid secretion decreased by 43-77% whereas only slight variations were observed in the different cell population densities within the gastric mucosa. In addition, no variation in gastrin levels was observed. Transgenics were protected against indomethacin-induced ulcer formation, with lesions diminishing by 45 to 64% compared to controls. These results indicate that endogenous apoA-IV expression can regulate gastric acid secretion and ulcer development.
Assuntos
Apolipoproteínas A/genética , Ácido Gástrico/metabolismo , Úlcera Gástrica/genética , Fatores Etários , Animais , Mucosa Gástrica/metabolismo , Gastrinas/metabolismo , Humanos , Indometacina/farmacologia , Camundongos , Camundongos Transgênicos , Úlcera Gástrica/metabolismo , Úlcera Gástrica/patologiaRESUMO
Rev-erb beta is an orphan receptor that binds as a homodimer or as a monomer to DNA. The solution structure of the non-palindromic 15 bp DNA duplex d(TAGAATGTAGGTCAG), the response element of Rev-erb beta for monomeric binding, was determined by 1H and 31P NMR, energy minimization with NMR-derived restraints for distances and NOE back-calculation methods. The refined final structures have the typical overall features of B-type DNA. However, titration of this 15 bp duplex with ReDBD, the DNA binding domain of Rev-erb beta, showed large shifts of imino protons and 31P signals, suggesting major conformational changes.
Assuntos
Proteínas de Ligação a DNA/química , Receptores Citoplasmáticos e Nucleares/química , Receptores dos Hormônios Tireóideos , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Simulação por Computador , DNA/química , Proteínas de Ligação a DNA/metabolismo , Dimerização , Hidrogênio , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular/métodos , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/metabolismo , Fósforo , Estrutura Secundária de Proteína , Receptores Citoplasmáticos e Nucleares/metabolismo , SoluçõesRESUMO
A simple chemical method for the synthesis of non-radioactive DNA probes is described: triazolyl-containing sequences were built by incorporation of 4-triazolylpyrimidin-2-ones instead of cytidines during oligodeoxyribonucleotide synthesis. The activating triazolyl groups were then displaced by a diamine which was further derivatized by a label, such as biotin. Synthesized DNA probes were oligonucleotides complementary to a cloned human antithrombin III DNA sequence. These probes, containing the same label at different positions of the sequence, were hybridized to their target DNA immobilized on nitrocellulose. Their hybridization specificity and stability were studied. Hybrid detection was performed either colorimetrically by the streptavidin-alkaline phosphatase-based system or by autoradiography after 5'-32P labeling of the probes: 15 fmol (0.05 microgram) of complementary sequence could be visualized in the two cases.
Assuntos
Antitrombina III/genética , Sondas de DNA/síntese química , Hibridização de Ácido Nucleico , Sequência de Bases , Biotina , Desnaturação de Ácido NucleicoRESUMO
Tacaribe arenavirus S RNA was cloned and analysis of its nucleotide sequence revealed two open reading frames of significant size, one in the virus-sense strand, the other in the virus-complementary strand. The predicted amino acid sequences of the two reading frames were compared with the predicted primary structures of the nucleoprotein (N) and glycoprotein precursor (GPC) of LCM, Pichinde and Lassa viruses. The results indicated a high degree of homology between the proteins of similar properties. It was also found that in Tacaribe virus-infected cells a subgenomic viral-sense GPC RNA and a subgenomic viral-complementary N RNA are synthesized in addition to the full length viral (v) RNA and viral complementary (vc) RNAs. These results support the conclusion that in Tacaribe virus--as in Pichinde and lymphocytic choriomeningitis arenavirus-S RNA encodes the viral N and GPC proteins and has an 'ambisense' coding strategy. Analysis of the S-derived RNA species at early times post-infection in cells incubated with or without inhibitors of protein synthesis indicated that for primary transcription of the N mRNA, protein synthesis is not required; whereas synthesis of the vc RNA, GPC mRNA and v RNA does require protein synthesis to take place.