RESUMEN
BACKGROUND: Hepatitis G Virus (HGV)/GB Virus-C (GBV-C) is a newly discovered RNA virus. Nucleotide sequence comparison and phylogenetic studies of the 5' untranslated region (5'UTR) within the viral genome have identified at least three different types which have provisionally been classified as type 1 (West African origin), type 2 (North American origin) and type 3 (Asian origin). METHODS AND RESULTS: The products of RT-PCR were sequenced by using blood donors and patients infected with HGV/GBV-C in Australia, Papua New Guinea and the Solomon Islands to investigate the genotype distribution in this area of the world. All the Australian isolates showed strong sequence homology with type 2, while the Papua New Guinea and Solomon Islands sequences were more closely related, but differ from type 3, which has previously been reported from isolates studied within Asia. CONCLUSIONS: Phylogenetic analysis suggests that these latter sequences are either a new HGV/GBV-C Pacific type or a subtype of the Asian type RNA virus. Isolates homologous with type 1 were not identified in these population groups.
Asunto(s)
Flaviviridae/genética , Hepatitis Viral Humana/genética , Australia/epidemiología , Variación Genética , Genoma Viral , Genotipo , Humanos , Melanesia/epidemiología , Papúa Nueva Guinea/epidemiología , Filogenia , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Análisis de Secuencia de ARNRESUMEN
BACKGROUND: The epidemiology and disease association for the GB virus type C (GBV-C) or hepatitis G virus (HGV) are poorly understood. STUDY DESIGN AND METHODS: This study describes the exposure rates to GBV-C/HGV in diverse Australian population groups by testing for current infection and evidence of past infection with a reverse transcriptase polymerase chain reaction and an anti-E2 enzyme-linked immunosorbent assay, respectively. Subjects included volunteer blood donors, hepatitis C antibody (anti-HCV)-positive donors, children, hemodialysis patients, pregnant women attending a prenatal clinic, injecting drug users (IVDUs), and adult hemophiliacs. RESULTS: Combined GBV-C RNA and E2 antibody prevalence was 6.5 percent (6/93) in children, 13.3 percent (75/565) in blood donors, 14 percent (14/99) in pregnant women, 22.5 percent (18/80) in hemodialysis patients, 80 percent (56/70) in anti-HCV-positive donors, 88.6 percent (31/35) in IVDUs, and 85.7 percent (54/63) in adult hemophiliacs. Children had the lowest antibody rate, 1.1 percent, whereas the rate was 10.8 percent for blood donors and rose to 45.7 percent for IVDUs, 57.1 percent for anti-HCV-positive donors, and 74.6 percent for hemophiliacs. In contrast, current infection rates were comparable for children, blood donors, and pregnant women (5.4, 2.6, and 6%, respectively), rising to 11.1 percent for hemophiliacs, 24.3 percent for anti-HCV-positive donors, and 48.6 percent for IVDUs. Ten of 12 blood donors had persistent viremia, while 2 had recent infections, 1 with apparent resolution. CONCLUSION: Exposure to GBV-C can commence at an early age, although ongoing exposure may also occur among adults with no apparent risk factors. GBV-C RNA positivity was not associated with abnormal plasma alanine aminotransferase levels among blood donors.
Asunto(s)
Flaviviridae , Hepatitis Viral Humana/epidemiología , Adulto , Anticuerpos Antivirales/sangre , Australia/epidemiología , Donantes de Sangre , Niño , Preescolar , Femenino , Flaviviridae/genética , Flaviviridae/inmunología , Hepatitis Viral Humana/virología , Humanos , Lactante , Masculino , Reacción en Cadena de la Polimerasa , Embarazo , ARN Viral/sangre , ADN Polimerasa Dirigida por ARN , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
We have sequenced a genomic DNA fragment containing the promoter and 5'-flanking region of the alpha 2MR/LRP. A cluster of five Sp1 sites situated over 600 base pairs away from the putative transcription start site doubles the activity of the promoter. A similar increase in activity was observed when this region was replaced by the SV40 enhancer, but the presence of both the cluster of Sp1 sites and SV40 enhancer gave no more transcription than either region alone. Within the previously described promoter region we have shown that only the most proximal Sp1 binding site influences transcription in CHO cells. The Sp 1 site situated 346 bp upstream of the putative transcription start site and previously described DNAse protection footprints had no effect on promoter activity in CHO cells. We also describe an NRF-1 binding site situated 143 bp upstream of the putative transcription start site. Deletion of the central 4 bp of this site caused a 60% decrease in transcription. No sterol regulatory (SRE-1) sites, used in the LDL receptor promoter for control of expression by cholesterol, were found in the alpha 2MR/LRP 5'-flanking region. However, one SRE-1 site was identified in the 5'-untranslated region of alpha 2MR/LRP.
Asunto(s)
Elementos de Facilitación Genéticos , Receptores Inmunológicos/genética , Factor de Transcripción Sp1/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Secuencia de Consenso , Proteínas de Unión al ADN/metabolismo , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Nucleares/metabolismo , Oligodesoxirribonucleótidos/química , ARN Mensajero/genética , Ratas , Alineación de Secuencia , Eliminación de Secuencia , Homología de Secuencia de Ácido Nucleico , Relación Estructura-Actividad , Transcripción GenéticaRESUMEN
The housekeeping enzyme 5-aminolevulinate synthase (ALAS) regulates the supply of heme for respiratory cytochromes. Here we report on the isolation of a genomic clone for the rat ALAS gene. The 5'-flanking region was fused to the chloramphenicol acetyltransferase gene and transient expression analysis revealed the presence of both positive and negative cis-acting sequences. Expression was substantially increased by the inclusion of the first intron located in the 5'-untranslated region. Sequence analysis of the promoter identified two elements at positions -59 and -88 bp with strong similarity to the binding site for nuclear respiratory factor 1 (NRF-1). Gel shift analysis revealed that both NRF-1 elements formed nucleoprotein complexes which could be abolished by an authentic NRF-1 oligomer. Mutagenesis of each NRF-1 motif in the ALAS promoter gave substantially lowered levels of chloramphenicol acetyltransferase expression, whereas mutagenesis of both NRF-1 motifs resulted in the almost complete loss of expression. These results establish that the NRF-1 motifs in the ALAS promoter are critical for promoter activity. NRF-1 binding sites have been identified in the promoters of several nuclear genes encoding mitochondrial proteins concerned with oxidative phosphorylation. The present studies suggest that NRF-1 may co-ordinate the supply of mitochondrial heme with the synthesis of respiratory cytochromes by regulating expression of ALAS. In erythroid cells, NRF-1 may be less important for controlling heme levels since an erythroid ALAS gene is strongly expressed and the promoter for this gene apparently lacks NRF-1 binding sites.
Asunto(s)
5-Aminolevulinato Sintetasa/genética , Factores Biológicos/metabolismo , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos Nucleicos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Factores Biológicos/genética , Células CHO , Línea Celular , Cloranfenicol O-Acetiltransferasa/genética , Cloranfenicol O-Acetiltransferasa/metabolismo , Clonación Molecular , Cricetinae , ADN/genética , Biblioteca Genómica , Humanos , Hígado/enzimología , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos , Plásmidos , Poli A/genética , Poli A/aislamiento & purificación , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Mapeo Restrictivo , Transfección , Células Tumorales CultivadasRESUMEN
In this paper, we have constructed deletion and deletion-insertion mutations of the chicken 5-aminolevulinate synthase 5' flanking region and examined the expression of these constructs in microinjected Xenopus oocytes. Utilising this assay, we have delimited the boundary of the 5' flanking region required for expression to be 80 bp upstream from the transcription initiation site. A second major focus of this study has been to define the role of known putative cis-acting sequences in regulating 5-aminolevulinate synthase gene expression. Expression of the insertion-deletion mutants demonstrated that only a TATA box at position -28, and a single GC box at position -78 was necessary for expression of the 5-aminolevulinate synthase gene in Xenopus oocytes. This result is unusual in view of the current state of knowledge of the function of cis-acting sequence elements in transcriptional regulation.
Asunto(s)
5-Aminolevulinato Sintetasa/genética , ADN/genética , Regulación de la Expresión Génica , Oocitos/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Animales , Secuencia de Bases , Pollos , Datos de Secuencia Molecular , Mutación , Regiones Promotoras Genéticas , Transcripción Genética , Transformación Genética , Xenopus laevisRESUMEN
The gene for 5-aminolevulinate synthase (ALAS) has been mapped to 3pter-3q13.2 by Southern blot hybridization analysis of a mouse/human hybrid cell panel. In situ hybridization maps the gene to 3p21, distal to the common fragile site at 3p14.2 (FRA3B). The mapping of this gene to an autosome makes it improbable that it is the site of the primary defect in X-linked sideroblastic anemia.
Asunto(s)
5-Aminolevulinato Sintetasa/genética , Anemia Sideroblástica/genética , Cromosomas Humanos Par 3 , Mapeo Cromosómico , Clonación Molecular , Humanos , Hibridación de Ácido NucleicoRESUMEN
cDNA clones for rat liver 5-aminolevulinate synthase have been isolated and used to examine mRNA levels in different rat tissues. Northern hybridization analysis of total RNA from various rat tissues showed the presence of a single 5-aminolevulinate synthase mRNA species of estimated length 2.3 kilobases. Primer extension and RNase mapping studies indicated that the mRNA is identical in all tissues. Highest basal levels were seen in liver and heart. Administration of hemin to rats reduced the basal level of this mRNA only in liver but the heme precursor, 5-aminolevulinate (or its methyl ester), repressed the basal levels in liver, kidney, heart, testis, and brain. The drug 2-allyl-2-isopropylacetamide increased the mRNA level in liver and kidney only while human chorionic gonadotropin hormone elevated the level in testis. Administration of the heme precursor 5-aminolevulinate prevented these inductions. Nuclear transcriptional run-off experiments in liver cell nuclei showed that 2-allyl-2-isopropylacetamide and 5-aminolevulinate exert their effect by altering the rate of transcription of the 5-aminolevulinate synthase gene. The results indicate that a single 5-aminolevulinate synthase mRNA is expressed in all tissues and that its transcription is negatively regulated by heme.
Asunto(s)
5-Aminolevulinato Sintetasa/genética , ARN Mensajero/metabolismo , Animales , Secuencia de Bases , Codón , ADN/análisis , Hemina/farmacología , Masculino , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Ratas , Ratas Endogámicas , Ribonucleasas/metabolismo , Distribución Tisular , Transcripción GenéticaRESUMEN
Reports to date have led to the conclusion that there are isozymes for 5-aminolevulinate synthase in the liver and erythroid tissue of chicken. Indeed, the existence of a multigene family for chicken 5-aminolevulinate synthase has been proposed. We find no evidence to support these proposals. In this work we show that 5-aminolevulinate synthase mRNA from chicken liver and reticulocytes is identical as determined by RNase mapping and primer extension studies and that the 5-aminolevulinate synthase protein from these tissues is the same size as judged by immunoblot analysis. We also show that a single mRNA species for 5-aminolevulinate synthase is present in chicken liver, reticulocytes, brain, and heart and an avian erythroblastosis virus-transformed chicken erythroblast cell line. Southern analysis shows the presence of only one gene copy for 5-aminolevulinate synthase in the chicken haploid genome. Overall, these results lead to the conclusion that in chickens 5-aminolevulinate synthase is encoded by a unique gene and is expressed as a single mRNA species in all tissues.
Asunto(s)
5-Aminolevulinato Sintetasa/genética , Hígado/análisis , ARN Mensajero/análisis , Reticulocitos/análisis , Alpharetrovirus , Animales , Química Encefálica , Línea Celular , Transformación Celular Viral , Pollos , ADN/genética , ADN Recombinante , Electroforesis en Gel de Poliacrilamida , Eritroblastos/análisis , Pruebas Inmunológicas , Miocardio/análisis , Hibridación de Ácido Nucleico , RibonucleasasRESUMEN
5-Aminolevulinate synthase, the first and rate-controlling enzyme of heme biosynthesis, is regulated in the liver by the end-product heme. To study this negative control mechanism, we have isolated the chicken gene for ALA-synthase and determined the nucleotide sequence. The structural gene is 6.9 kb long and contains 10 exons. The transcriptional start site for ALA-synthase was determined by primer extension analysis. A fragment of 291 bp from the 5' flanking region including 34 bp of the first exon shows promoter activity when introduced upstream of a chicken histone H2B gene and injected into the nuclei of Xenopus laevis oocytes.
Asunto(s)
5-Aminolevulinato Sintetasa/genética , Pollos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Embrión de Pollo , Mapeo Cromosómico , Enzimas de Restricción del ADN , Regulación de la Expresión Génica/efectos de los fármacos , Genes , Ligamiento Genético , Mitocondrias Hepáticas/enzimología , Regiones Promotoras GenéticasRESUMEN
The proposed mechanism by which hepatic ALV-synthase mitochondrial levels are regulated is outlined in Fig. 2. ALV-synthase catalyzes the first and rate-limiting step in the heme pathway and is normally present in low amounts. A cytosolic, regulatory free heme pool tightly controls the amount of ALV-synthase in two ways. In the primary mechanism of regulation, heme is proposed to inhibit the synthesis of ALV-synthase mRNA. Most likely this would be mediated through the action of specific heme-binding protein(s) which recognize regulatory control regions of the ALV-synthase gene. Gene activity therefore is significantly repressed most of the time. When there is an increased demand for heme by newly synthesized cellular hemoproteins, the free heme pool is reduced, leading to a derepression of ALV-synthase mRNA synthesis. Once the need for increased heme synthesis is satisfied, inhibitory heme levels build up again. When drugs such as phenobarbital are administered to animals, there is a rapid induction in the liver of both cytochrome P-450 and ALV-synthase. It is proposed that the heme pool governing ALV-synthase levels is lowered by the increased heme demand due to cytochrome P-450 apoprotein formation. The primary event in the drug induction of ALV-synthase is therefore the increased synthesis of cytochrome P-450 apoprotein. However, the mechanism by which this occurs is unknown, although drugs do increase the synthesis of mRNA for cytochrome P-450 (Fig. 2). (There is evidence that for the aromatic hydrocarbons a specific cytosolic receptor exists.) In the acute hepatic porphyria diseases, uncontrolled synthesis of hepatic ALV-synthase occurs. The various forms are characterized by reduced levels of one of the heme pathway enzymes other than ALV-synthase. Attacks of the disease are commonly precipitated by drugs which induce cytochrome P-450, and the uncontrolled accumulation of ALV-synthase which accompanies these attacks results from the combined action of the block in the heme pathway and the increased cytochrome P-450 levels. A major challenge which now exists is to understand at the molecular level how the genes for ALV-synthase and cytochrome P-450 are regulated in the liver and other tissues.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
5-Aminolevulinato Sintetasa/metabolismo , Animales , Citosol/enzimología , Hemo/metabolismo , Humanos , Hígado/enzimología , Mitocondrias/enzimología , Mitocondrias Hepáticas/enzimología , Porfirias/enzimología , Reticulocitos/enzimologíaRESUMEN
Chick embryo liver mitochondrial matrix protein, 5-aminolaevulinate synthase, is synthesised initially as a larger cytosolic precursor. In this report we present the complete nucleotide sequence of a cDNA clone coding for the precursor together with corresponding confirmatory amino acid sequence of peptides derived from purified mature mitochondrial enzyme. The deduced amino acid sequence shows that the precursor consists of mature enzyme of 579 amino acids and an N-terminal extension of 56 amino acids. The latter presequence is highly basic in character as found with other mitochondrial preproteins.
Asunto(s)
5-Aminolevulinato Sintetasa/biosíntesis , Precursores Enzimáticos/biosíntesis , Mitocondrias Hepáticas/enzimología , 5-Aminolevulinato Sintetasa/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Embrión de Pollo , Clonación Molecular , ADN , Precursores Enzimáticos/genética , Fragmentos de Péptidos/análisisRESUMEN
The structure of chick embryo liver 5-aminolaevulinate synthase has been examined by electron microscopic studies using negative staining. From the different projections of the enzyme particles observed in electron micrographs, a model for the enzyme molecule has been proposed. In this model, an enzyme molecule consists of two curved and identical subunits associated in opposite polarities. From the dimensions of an enzyme molecule subunit measured from electron micrographs, the relative molecular mass of each subunit is estimated to be 70 000.
Asunto(s)
5-Aminolevulinato Sintetasa/análisis , Hígado/enzimología , Animales , Embrión de Pollo , Hígado/ultraestructura , Microscopía Electrónica , Modelos EstructuralesRESUMEN
Hepatic 5-aminolevulinate synthase was induced in chick embryos by administration of the porphyrinogenic drugs 2-allyl-2-isopropylacetamide and 3,5-diethoxycarbonyl-1,4-dihydrocollidine. A cDNA library was constructed from drug-induced liver mRNA and clones containing sequences coding for 5-aminolevulinate synthase were identified by hybrid-selected translation. The identity of these clones was confirmed by comparing DNA sequence data with the amino acid sequence of peptides from purified 5-aminolevulinate synthase. From Northern blot analysis the size of the mRNA for 5-aminolevulinate synthase was estimated to be 2800 base pairs, approximately 600 base pairs more than that required to code for the primary translation product of relative molecular mass 74000.
Asunto(s)
5-Aminolevulinato Sintetasa/genética , Clonación Molecular , Hígado/enzimología , 5-Aminolevulinato Sintetasa/biosíntesis , Alilisopropilacetamida/farmacología , Secuencia de Aminoácidos , Animales , Embrión de Pollo , ADN/aislamiento & purificación , Dicarbetoxidihidrocolidina/farmacología , Inducción Enzimática/efectos de los fármacos , Hibridación de Ácido Nucleico , Polirribosomas/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , ARN Mensajero/aislamiento & purificaciónRESUMEN
Pb2+ activated native chick-embryo liver mitochondrial delta-aminoaevulinate synthase (EC 2.3.1.37). This result contradicted with the inhibitory effect observed by earlier workers who used degraded enzyme preparations. Enzyme activation was biphasic. An initial activation phase was observed with Pb2+ concentrations up to 200 microM, and a secondary phase with concentrations from 200 microM to at least 2mM. Maximum primary activation was 2.5-fold at 200 microM-Pb2+, with a further 2-fold activation observed at 2mM-Pb2+. Primary activation was not affected by a 10-fold molar excess of dithioerythritol, but the secondary activation was abolished by dithioerythritol. Secondary-phase activation was lost upon increasing time of incubation of the enzyme with Pb2+. The implications of these findings are discussed with reference to lead poisoning and the mechanism of delta-aminolaevulinate synthase.
Asunto(s)
5-Aminolevulinato Sintetasa/metabolismo , Plomo/farmacología , Mitocondrias Hepáticas/enzimología , Animales , Embrión de Pollo , Ditioeritritol/farmacología , Activación Enzimática/efectos de los fármacos , Factores de TiempoRESUMEN
We have examined the effect of heme on the activity of native delta-aminolevulinate synthase isolated from drug-induced chick embryo liver mitochondria. The enzyme was not inhibited by concentrations of heme up to 1 mM and this finding makes it improbable that heme acts physiologically to control mitochondrial delta-aminolevulinate synthase activity.
Asunto(s)
5-Aminolevulinato Sintetasa/metabolismo , Hemo/farmacología , Mitocondrias Hepáticas/enzimología , Animales , Embrión de Pollo , Activación Enzimática , Sulfato de Magnesio/farmacología , Cloruro de Sodio/farmacologíaRESUMEN
Pulse labelling studies in chick embryo livers show that hemin prevents the transfer of drug induced pre-delta-aminolevulinate synthase from the cytosol into the mitochondria, leading to an accumulation of precursor in the cytosol. No effect of hemin was observed on the transfer of pre-pyruvate carboxylase into mitochondria. These results eliminated a general toxic effect of hemin on mitochondrial import of proteins and are consistent with the view that hemin specifically inhibits the transfer of ALA synthase.
Asunto(s)
5-Aminolevulinato Sintetasa/genética , Precursores Enzimáticos/genética , Hemo/farmacología , Mitocondrias Hepáticas/enzimología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Animales , Embrión de Pollo , Citosol/enzimología , Hígado/enzimología , Peso Molecular , Piruvato Carboxilasa/genéticaRESUMEN
The induction of cytochrome P450 in chick embryo liver has been studied using three different porphyrinogenic drugs, 2-allyl-2-isopropylacetamide, 3,5-diethoxycarbonyl-1,4-dihydrocollidine and phenobarbital. Pulse-labelling studies have shown that for each drug the cytochrome P450 synthesized either in ovo or in a wheat germ translation system reacted immunologically with antibody raised against the purified 2-allyl-2-isopropylacetamide-induced enzyme (Mr = 50000). To investigate whether this is due to the three drugs inducing the same protein or different proteins with common immunological determinants, nucleic acid hybridization studies have been carried out using a recently characterised 2-allyl-2-isopropylacetamide-induced cytochrome P450 cloned cDNA probe [Brooker, J. D. et al. (1982) Eur. J. Biochem. 129, 325-333]. It has been shown that the mRNA induced by each drug hybridizes with this probe and all are of similar size. The melting profile of the mRNA . cDNA hybrids indicates that the mRNAs induced by the three drugs have at least 98% homology with the cDNA probe. Restriction endonuclease digestions of total chick embryo genomal DNA and a chick cytochrome P450 genomal clone indicates that the cytochrome P450 gene homologous with the cDNA probe is represented in the genome only once. These results strongly suggest that the three drugs cause increased levels of the same cytochrome P450 mRNA, possibly due to enhanced expression of the same gene. Results are also presented which show that other cytochrome-P450-inducing drugs, 3-methylcholanthrene, beta-naphthoflavone or pregnenolone-16 alpha-carbonitrile do not increase the level of the 2-allyl-2-isopropylacetamide-inducible mRNA but rather reduce it to a level which was lower than that of the untreated controls.
Asunto(s)
Acetamidas/farmacología , Alilisopropilacetamida/farmacología , Sistema Enzimático del Citocromo P-450/biosíntesis , Dicarbetoxidihidrocolidina/farmacología , Hígado/enzimología , Fenobarbital/farmacología , Piridinas/farmacología , ARN Mensajero/metabolismo , Animales , Fenómenos Químicos , Química , Embrión de Pollo , ADN , Inducción Enzimática/efectos de los fármacos , Hibridación de Ácido NucleicoRESUMEN
Following the recent demonstration [Borthwick, I.A., Srivastava, G., Brooker, J.D., May, B.K. and Elliott, W.H. (1982) Eur. J. Biochem. in press] that chick embryo liver mitochondrial delta-aminolevulinate synthase has a minimum molecular weight of 68,000 (rather than the hitherto accepted value of 49,000), we have shown that the primary translation product of delta-aminolevulinate synthase mRNA is a protein of molecular weight 74,000. This protein has for the first time been shown to occur in the cytosol fraction of drug-treated chick embryo livers. This form does not occur in mitochondria nor does the smaller mitochondrial form occur in the cytosol. It is concluded that the 74,000 molecular weight protein is a precursor which is processed during transport into the mitochondria. In vivo labelling experiments are consistent with this conclusion.