RESUMEN
Localization of cytochrome P-450 on various membrane fractions of rat liver cells was studied by direct immunoelectron microscopy using ferritin-conjugated antibody to the cytochrome. The outer surfaces of almost all the microsomal vesicles were labeled with ferritin particles. The distribution of the particles on each microsomal vesicle was usually heterogeneous, indicating clustering of the cytochrome, and phenobarbital treatment markedly increased the labeled regions of the microsomal membranes. The outer nuclear envelopes were also labeled with ferritin particles, while on the surface of other membrane structures such as Golgi complexes, outer mitochondrial membranes and plasma membranes the labeling was scanty and at the control level. The present observation indicates that cytochrome P-450 molecules are localized exclusively on endoplasmic reticulum membranes and outer nuclear envelopes where they are probably distributed not uniformly but heterogeneously, forming clusters or patches. The physiological significance of such microheterogeneity in the distribution of the cytochrome on endoplasmic reticulum membranes is discussed.
Asunto(s)
Sistema Enzimático del Citocromo P-450/aislamiento & purificación , Membranas Intracelulares/análisis , Hígado/análisis , Microsomas Hepáticos/análisis , Animales , Reacciones Antígeno-Anticuerpo , Membrana Celular/análisis , Retículo Endoplásmico/análisis , Aparato de Golgi/análisis , Masculino , Microscopía Electrónica , Mitocondrias Hepáticas/análisis , Membrana Nuclear/análisis , Ratas , Fracciones SubcelularesRESUMEN
Intracellular sites of synthesis of cytochrome P-450 and the subsequent incorporation of it into membrane structures of the endoplasmic reticulum (ER) in rat hepatocytes have been studied using an antibody monospecific for phenobarbital-inducible cytochrome P-450. The cytochrome is synthesized mainly on the "tightly bound" type of membrane-bound ribosomes whose release from the membrane requires treatment with puromycin in a high salt buffer (500 mM KCI, 5mM MgCl2, and 50 mM Tris-HCL [pH 7.5]). Subsequently the cytochrome is incorporated directly into the rough ER membranes with its major part exposed to the outer surface to the membrane and accessible to proteolytic enzymes added externally. The newly synthesized molecules, which appeared first in the rough membrane, are translocated to the smooth membrane, and are then distributed evenly between the two types of microsomeal membranes in approximately 1 h. Administration of cycloheximide, an inhibitor of protein biosynthesis, did not significantly inhibit the transfer of the enzyme from the rough to the smooth ER. It is suggested, therefore, that the translocation of the newly synthesized cythochrome P-450 between the rough and smooth microsomes is mainly due to the lateral movement of the molecules in the plane of the membranes rather than to the attachment and detachment of the ribosomes on the microsomal membranes after the ribosomal cycle for protein synthesis.
Asunto(s)
Sistema Enzimático del Citocromo P-450/biosíntesis , Microsomas Hepáticos/metabolismo , Proteínas Ribosómicas/biosíntesis , Animales , Sitios de Unión , Sistema Enzimático del Citocromo P-450/inmunología , Sistema Enzimático del Citocromo P-450/aislamiento & purificación , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Membranas Intracelulares/metabolismo , Masculino , Microsomas Hepáticos/ultraestructura , Puromicina/farmacología , Ratas , Propiedades de SuperficieRESUMEN
Steroid 21-hydroxylase deficiency is a major cause of congenital adrenal hyperplasia and is caused by genetic impairment of this enzyme. Since approximately 80% of cases are caused by point mutations of the CYP21B (CYP21A2) gene, whereas the remaining 20% are due to deletion of this gene, we used the polymerase chain reaction single strand conformation polymorphism technique for rapid and accurate diagnosis of this disease. Of 23 patients examined, 1 had a hemizygous CYP21B gene. 18 patient's genes localized their harmful mutations or deletion on both the alleles, while 4 of them found their causative mutations on one of the two alleles, and 1 failed to find any responsible mutation. All the mutations (four nucleotide substitutions) detected are also found in the CYP21A (CYP21A1) pseudogene. A mutation at the intron 2 site is most prevalent in both salt-wasting and simple virilizing forms of the disease, and accounts for 37% of the patient's genes (17/46). Pedigree analysis of these mutations revealed that the mutations (at least four of them) occurred de novo at a considerable frequency on both the paternally and maternally inherited chromosomes. This result could explain occasional discordance of the diagnosis using HLA typing with the clinical symptoms.
Asunto(s)
Hiperplasia Suprarrenal Congénita , Hiperplasia Suprarrenal Congénita/diagnóstico , Hiperplasia Suprarrenal Congénita/genética , Mutación Puntual , Esteroide 21-Hidroxilasa/genética , Hiperplasia Suprarrenal Congénita/enzimología , Alelos , Secuencia de Bases , Cartilla de ADN , Exones/genética , Femenino , Heterocigoto , Humanos , Intrones/genética , Masculino , Datos de Secuencia Molecular , Mutagénesis , Conformación de Ácido Nucleico , Linaje , Reacción en Cadena de la Polimerasa/métodos , Seudogenes , Análisis de Secuencia de ADNRESUMEN
The expression of the fast type of myosin alkali light chain 1 is induced during the differentiation of muscle cells. To study the mechanism of its gene regulation, we joined the sequence of the 5'-flanking and upstream region of the chicken myosin alkali light-chain gene to the structural gene for chloramphenicol acetyltransferase (CAT). The fusion gene was introduced either into quail myoblasts transformed by a temperature-sensitive mutant of Rous sarcoma virus (tsNY68) or into chicken myoblasts, and the transiently expressed CAT activity was assayed after the differentiation of the myoblasts. From the experiments with the external and internal deletion mutants of the fusion gene, the cis-acting regulatory region responsible for the enhanced expression of the CAT activity in response to the cell differentiation was found to be localized at 2 kilobases upstream of the transcription initiation site. This region of 160 nucleotides contained two pairs of short sequences worthy of note, a direct repeat of 12 nucleotides, and an inverted repeat of 8 nucleotides. The nucleotide sequences of the 5'-flanking sequence up to nucleotide -3381 were determined and compared with those of the upstream activating elements of actin genes.
Asunto(s)
Regulación de la Expresión Génica , Miosinas/genética , Animales , Secuencia de Bases , Línea Celular Transformada , Células Cultivadas , Pollos , Datos de Secuencia Molecular , Mutación , Secuencias Reguladoras de Ácidos Nucleicos , Transcripción Genética , TransfecciónRESUMEN
A novel cis-acting regulatory element (designated BTE for basic transcription element) was found in the region proximal to the TATA sequence of the P-450c gene by the use of deletion mutations. This DNA element is considered to be involved in the basic transcription of the gene and does not show distinct enhancer activity in itself. Together with the XRE sequence (A. Fujisawa-Sehara, K. Sogawa, M. Yamane, and Y. Fujii-Kuriyama, Nucleic Acids Res. 15:4179-4191, 1987), however, this sequence is required for a high inducible expression of the P-450c gene in response to xenobiotic inducers. The BTE sequence contained the GC box consensus sequence and half of the NF-1-binding consensus or CAT box sequence, but their synthetic oligonucleotides, used as competitors in the gel mobility shift assays, did not compete with the BTE sequence for the binding protein, suggesting that the BTE sequence functions as a different recognition sequence from that for Sp1 or NF-1. Analogous sequences to BTE are found in the region proximal to the TATA sequence of other genes, especially other P-450 genes with different modes of regulation, suggesting that the BTE sequence plays a common regulatory role in basic transcription of genes including a group of the P-450 superfamily. The ubiquitous distribution of nuclear factor(s) binding to this element supports this suggestion.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Regulación de la Expresión Génica , Genes , Regiones Promotoras Genéticas , Animales , Secuencia de Bases , Línea Celular , Cloranfenicol O-Acetiltransferasa/genética , Deleción Cromosómica , Datos de Secuencia Molecular , Mutación , Sondas de Oligonucleótidos , Ratas , Homología de Secuencia de Ácido Nucleico , TransfecciónRESUMEN
A dominant mutant of Hepa-1 cells, c31, expresses a repressor that prevents 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-dependent stimulation of Cyp1a1 transcription. The repressor acts via the xenobiotic-responsive elements (XREs), which are the DNA-binding sites for the aryl hydrocarbon (Ah) receptor-TCDD complex during transcriptional activation of the gene. High-salt nuclear extracts prepared from c31 cells grown with TCDD contained normal levels of the Ah receptor which bound the XRE with normal affinity, as judged by in vitro gel mobility shift assays. Furthermore, extracts prepared from these cells, grown either with or without TCDD, contained no novel XRE-binding proteins compared with extracts from wild-type Hepa-1 cells. However, in vivo genomic footprinting demonstrated that TCDD treatment leads to binding of the Ah receptor to the XREs in Hepa-1 but not mutant cells. This finding suggests that the repressor associates with the Ah receptor to prevent its binding to the XREs and that high-salt treatment either causes dissociation of the receptor/repressor complex or fails to extract the repressor from nuclei. The results underscore the importance of using both in vivo and in vitro assays for analyzing DNA-protein interactions.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Dibenzodioxinas Policloradas/farmacología , Receptores de Droga/metabolismo , Proteínas Represoras/metabolismo , Transcripción Genética/efectos de los fármacos , Animales , Secuencia de Bases , Sitios de Unión , Línea Celular , Quimera , Cloranfenicol O-Acetiltransferasa/genética , Cloranfenicol O-Acetiltransferasa/metabolismo , Sistema Enzimático del Citocromo P-450/biosíntesis , Sistema Enzimático del Citocromo P-450/metabolismo , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Inducción Enzimática , Represión Enzimática , Genes Dominantes , Neoplasias Hepáticas Experimentales , Ratones , Datos de Secuencia Molecular , Mutación , Dibenzodioxinas Policloradas/metabolismo , Ratas , Receptores de Hidrocarburo de Aril , Proteínas Recombinantes de Fusión/metabolismo , Mapeo Restrictivo , TransfecciónRESUMEN
From a cDNA library of mouse skeletal muscle, we have isolated mouse Sim1 (mSim1) cDNA encoding a polypeptide of 765 amino acids with striking amino acid identify in basic helix-loop-helix (89% identify) and PAS (89 % identify) domains to previously identified mSim2, although the carboxy-terminal third of the molecule did not show any similarity to mSim2 or Drosophila Sim (dSim). Yeast two-hybrid analysis and coimmunoprecipitation experiments demonstrated that both of the mSim gene products interacted with Arnt even more efficiently than AhR, a natural partner of Arnt, suggesting a functional cooperativity with Arnt. In sharp contrast with dSim having transcriptional-enhancing activity in the carboxy-terminal region, the two mSims possessed a repressive activity toward Arnt in the heterodimer complex. This is the first example of bHLH-PAS proteins with transrepressor activity, although some genetic data suggest that dSim plays a repressive role in gene expression (Z. Chang, D. Price, S. Bockheim, M. J. Boedigheimer, R. Smith, and A. Laughon, Dev. Biol. 160:315-322, 1993; D. M. Mellerick and M. Nirenberg, Dev. Biol. 171:306-316, 1995). Whole-mount in situ hybridization showed restricted and characteristic expression patterns of the two mSim mRNAs in various tissues and organs during embryogenesis, such as those for the somite, the nephrogenic cord, and the mesencephalon (for mSim1) and those for the diencephalon, branchial arches, and limbs (for mSim2). From sequence similarity and chromosomal localization, it is concluded that mSim2 is an ortholog of hSim2, which is proposed to be a candidate gene responsible for Down's syndrome. The sites of mSim2 expression showed an overlap with the affected regions of the syndrome, further strengthening involvement of mSim2 in Down's syndrome.
Asunto(s)
Mapeo Cromosómico , Regulación del Desarrollo de la Expresión Génica , Familia de Multigenes , Músculo Esquelético/metabolismo , Proteínas Represoras/biosíntesis , Secuencia de Aminoácidos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Encéfalo/embriología , Encéfalo/metabolismo , Células COS , Línea Celular , Clonación Molecular , Proteínas de Unión al ADN/química , Drosophila , Proteínas de Drosophila , Desarrollo Embrionario y Fetal , Biblioteca de Genes , Secuencias Hélice-Asa-Hélice , Humanos , Hibridación Fluorescente in Situ , Cariotipificación , Ratones , Datos de Secuencia Molecular , Músculo Esquelético/embriología , Proteínas Nucleares/química , Sondas ARN , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes/biosíntesis , Proteínas Represoras/química , Homología de Secuencia de AminoácidoRESUMEN
We isolated mouse cDNA clones (Arnt2) that are highly similar to but distinct from the aryl hydrocarbon receptor (AhR) nuclear translocator (Arnt). The composite cDNA covered a 2,443-bp sequence consisting of a putative 2,136-bp open reading frame encoding a polypeptide of 712 amino acids. The predicted Arnt2 polypeptide carries a characteristic basic helix-loop-helix (bHLH)/PAS motif in its N-terminal region with close similarity (81% identity) to that of mouse Arnt and has an overall sequence identity of 57% with Arnt. Biochemical properties and interaction of Arnt2 with other bHLH/PAS proteins were investigated by coimmunoprecipitation assays, gel mobility shift assays, and the yeast two-hybrid system. Arnt2 interacted with AhR and mouse Sim as efficiently as Arnt, and the Arnt2-AhR complex recognized and bound specifically the xenobiotic responsive element (XRE) sequence. Expression of Arnt2 successfully rescued XRE-driven reporter gene activity in the Arnt-defective c4 mutant of Hepa-1 cells. RNA blot analysis revealed that expression of Arnt2 mRNA was restricted to the brains and kidneys of adult mice, while Arnt mRNA was expressed ubiquitously. In addition, whole-mount in situ hybridization of 9.5-day mouse embryos showed that Arnt2 mRNA was expressed in the dorsal neural tube and branchial arch 1, while Arnt transcripts were detected broadly in various tissues of mesodermal and endodermal origins. These results suggest that Arnt2 may play different roles from Arnt both in adult mice and in developing embryos. Finally, sequence comparison of the currently known bHLH/PAS proteins indicates a division into two phylogenetic groups: the Arnt group, containing Arnt, Arnt2, and Per, and the AhR group, consisting of AhR, Sim, and Hif-1alpha.
Asunto(s)
ADN Complementario/genética , Secuencias Hélice-Asa-Hélice , Receptores de Hidrocarburo de Aril/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Translocador Nuclear del Receptor de Aril Hidrocarburo , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Clonación Molecular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos , Genes Reporteros , Ratones , Datos de Secuencia Molecular , Receptores de Hidrocarburo de Aril/metabolismo , Homología de Secuencia de Aminoácido , Factores de Transcripción/metabolismoRESUMEN
Administration of 2-acetylaminofluorene to rats for 12 weeks induces hyperplastic nodules (HPNs) and later well-differentiated hepatocellular carcinomas (HCCs) in the liver. Total cellular proteins from normal liver, HPN, and HCC were analyzed by two-dimensional gel electrophoresis with a high resolution. Several hundred polypeptides were well resolved as seen by Coomassie blue staining, forming a reproducible and characteristic pattern for each tissue. The polypeptide patterns were very similar among normal liver, HPN, and HCC. Especially the proteins of HPN and HCC were almost indistinguishable. These neoplastic lesions, however, were clearly different from control liver in that a new spot p35-6.6 (designated by molecular weight X 10(-3) and pl) appeared, and five polypeptides, p57-6.9, p57-6.7, p26-6.9, p26-6.6, p26-6.4, increased dramatically in amount as compared with normal liver. These last three spots were found to be a new type of glutathione S-transferase as judged from the specific binding to the antibody. The same changes in polypeptide pattern were found in HCCs induced by other chemical carcinogens, diethylnitrosamine and 3'-methyl-4-dimethylaminoazobenzene, but not in regenerating and neonatal livers. Fetal liver showed a rather different pattern than adult liver, but only p26-6.6 was increased among the spots characteristic of HPN and HCC. Protein phosphorylation was also examined for these cells by incubating tissue slices with 32PO4. After alkali treatment of the gels to eliminate serines phosphorylation, several dozens of phosphoproteins were clearly detected. The patterns of the labeled spots were again very similar among control liver, HPN, and HCC. Only the intensity of a spot designated p57-6.6 increased markedly in both HPN and HCC. This spot was further resolved by an expanded pH gradient into four distinct spots, the major one of which contained phosphothreonine. Similar changes in phosphorylation were noted in hepatomas induced by diethylnitrosamine and 3'-methyl-4-dimethylaminoazobenzene but not in regenerating, fetal, and neonatal livers. These changes are discussed in terms of gene expression relevant to malignant transformation of hepatic cells.
Asunto(s)
Transformación Celular Neoplásica , Neoplasias Hepáticas Experimentales/patología , Hígado/patología , Proteínas de Neoplasias/análisis , Péptidos/análisis , 2-Acetilaminofluoreno , Envejecimiento , Animales , Dietilnitrosamina , Hígado/análisis , Hígado/crecimiento & desarrollo , Regeneración Hepática , Masculino , Metildimetilaminoazobenceno , Peso Molecular , Lesiones Precancerosas/patología , Ratas , Ratas EndogámicasRESUMEN
The relationship between methylation and expression of rat pepsinogen 1 (Pg1) genes was investigated in various tissues. On Northern blotting with a Pg1 complementary DNA probe, Pg1 mRNA was detected only in the glandular stomach of normal rats. Methylation analysis with Msp1/HpaII and Hha1 revealed tissue specific methylation patterns of Pg1 genes with less methylated in the stomach than in other normal tissues not expressing the genes. During stomach development, there was a progressive increase in the Pg1 mRNA level that almost coincided with change in the mucosal pepsinogen level and progressive demethylation after the onset of transcription. Thus, there was an inverse correlation between methylation and expression of Pg1 genes, suggesting a role of DNA methylation in Pg1 gene regulation during normal differentiation, although not its primary role in gene activation. There was no detectable Pg1 mRNA in either primary or transplanted stomach cancers induced by N-methyl-N'-nitro-N-nitrosoguanidine. The methylation patterns of Pg1 genes were different from those of normal tissues that expressed the gene and of those that did not and no simple correlation was observed between methylation and expression of Pg1 genes. This result is consistent with a previous finding that DNA methylation is deranged in tumor cells.
Asunto(s)
ADN/metabolismo , Mucosa Gástrica/metabolismo , Pepsinógenos/genética , Neoplasias Gástricas/metabolismo , Animales , Femenino , Regulación de la Expresión Génica , Masculino , Metilación , ARN Mensajero/análisis , Ratas , Ratas Endogámicas , Estómago/embriología , Activación TranscripcionalRESUMEN
The compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been shown recently to be carcinogenic, but little is currently known about the molecular mechanism of TCDD affecting cell proliferation and carcinogenesis. In this report, we demonstrate that TCDD suppresses the expression of the checkpoint protein, Mad2. Suppression of Mad2 was also observed in aryl hydrocarbon receptor-deficient mouse embryonic fibroblasts, suggesting that TCDD suppresses Mad2 by a novel TCDD receptor signaling mechanism. In addition, HeLa cells treated with TCDD failed to arrest in mitosis after nocodazole treatment. The Mad2 protein plays a significant role in accurate chromosome segregation in mitotic cells. Our data suggest that TCDD may increase chromosomal instability through the suppression of Mad2 expression.
Asunto(s)
Proteínas de Unión al Calcio/antagonistas & inhibidores , Carcinógenos Ambientales/toxicidad , Dibenzodioxinas Policloradas/toxicidad , Receptores de Hidrocarburo de Aril/fisiología , Animales , Proteínas de Unión al Calcio/biosíntesis , Ciclo Celular/efectos de los fármacos , Ciclo Celular/fisiología , Proteínas de Ciclo Celular , Cruzamientos Genéticos , Contaminantes Ambientales/toxicidad , Femenino , Células HeLa , Humanos , Proteínas Mad2 , Ratones , Ratones Endogámicos C57BL , Mitosis/efectos de los fármacos , Mitosis/fisiología , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismo , Proteínas RepresorasRESUMEN
By site-directed mutagenesis, we made several cytochrome P-450d (P-450d) mutants as follows: Asn310Phe (D13), Ile312Leu (D14), Glu318Asp (D15), Val320Ile (D16), Phe325Thr (D19), Asn310Phe,Ile312Leu (M6), Glu318Asp,Val320Ile (M7), Phe325Thr, Glu318Asp (M3). This region (Asn-310-Phe-325) is supposed to be located in the distal helix above the heme plane in P-450d, being conjectured from the structure of P-450cam. We studied Soret spectral changes of those mutants by adding several axial ligands such as aniline, pyridine, metyrapone, 2-phenylimidazole and 4-phenylimidazole. Binding constants (Kb) of aniline and pyridine to the single and double mutants were higher than those to the wild type by 2-10-times. The double mutations did not additively increase the Kb values compared with those to the single mutants. In contrast, Kb value (1.0.10(5) M-1) of metyrapone to the double mutant M3 was much higher than that (2.0.10(3) M-1) of the wild type and those of the single mutants, D15 (4.5.10(4) M-1) and D19 (1.6.10(4) M-1). The increased affinity of metyrapone to the mutant M3 may be attributed to an interaction of the hydrophobic group of metyrapone with nearby hydrophobic group(s) produced cooperatively by the double mutation of P-450d. Kb values of 2-phenylimidazole and 4-phenylimidazole to the mutant M3 were also the highest among those of the mutants and the wild type. Therefore, it was suggested that this region (from Asn-310 to Phe-325) must be located at the distal region of the heme moiety and form, at least, a substrate-binding region of membrane-bound P-450d.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Mutación , Secuencia de Aminoácidos , Compuestos de Anilina/metabolismo , Secuencia de Bases , Sistema Enzimático del Citocromo P-450/genética , ADN/genética , Imidazoles/metabolismo , Metirapona/metabolismo , Microsomas/enzimología , Piridinas/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Relación Estructura-ActividadRESUMEN
The metallothionein (MT) I and II genes were isolated from Chinese hamster cells and sequenced. The MT-II gene is located about 6 kb upstream of the MT-I gene and their arrangement is similar to those of the mouse and rat MT genes. The sequence of the Chinese hamster MT-I gene is highly homologous to those of the mouse and rat, particularly in their promoter regions of MT-I. However, the promoter region of MT-II has less homology with those of the mouse and rat due t to insertions and deletions. The MT-I and MT-II genes were equally amplified 4-8-times in the Cd-resistant Chinese hamster cells, suggesting that both genes are included in the same amplification unit. Cytogenetic analysis of Cd-resistant cells by in situ hybridization showed that they are randomly integrated into multiple sites on the chromosomes.
Asunto(s)
Cadmio/farmacología , Amplificación de Genes , Metalotioneína/genética , Muridae/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células Cultivadas , Mapeo Cromosómico , Clonación Molecular , Cricetinae , Cricetulus , ADN Complementario/genética , Resistencia a Medicamentos , Genoma , Hibridación in Situ , Ratones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Ratas , Homología de Secuencia de Ácido NucleicoRESUMEN
Low-temperature (6-40 K) electron spin resonance (ESR) spectra of cytochrome P-450d (P-450d) and its 17 mutants have been measured. The spectra of the wild-type and all mutant P-450ds showed signals at around g = 8, 3.7 and 1.7, while they didn't show any signal at around g = 2 up to 40 K. It was thus suggested that all of these P-450ds essentially take the ferric high-spin form. The g values of the proximal mutants were closer to those of the wild-type than those of the distal and aromatic mutants, suggesting that mutations at the distal and aromatic sites influence the electronic state of the heme more profoundly than those of the proximal site. The distal multiple mutants whose distal sequences are the same as those of the low-spin type P-450s such as rat P-450c, mouse P1-450 and P3-450 showed only high-spin ESR signals. Thus the spin state of P-450ds (the wild-type and all mutants) may not be solely due to specific characteristics of the distal site, but to the unique nature of the whole heme environment of P-450d. It is also suggested that the amino acids at the distal region of P-450d may be located close to the heme, so that the water molecule cannot bind to the heme, thus taking the high-spin state. Both the aromatic mutants showed rather large deviations of the g values from those of wild-type P-450d, suggesting that the aromatic region somehow interacts with the heme.
Asunto(s)
Sistema Enzimático del Citocromo P-450 , Animales , Monóxido de Carbono , Sistema Enzimático del Citocromo P-450/genética , Análisis Mutacional de ADN , Espectroscopía de Resonancia por Spin del Electrón , Hemo , Microsomas Hepáticos/enzimología , Ratas , Relación Estructura-ActividadRESUMEN
We have isolated and characterized two kinds of cDNA for the chicken cardiac myosin alkali light chain. The sequences of the two cDNAs are identical, except for a notable divergence in part of the 3' untranslated sequence. By analysis of isolated genomic clones, it was shown that the genomic sequences corresponding to the different sequences in the 3' untranslated regions of the two mRNAs were arranged within a limited part of a single stretch of DNA; also the two distinct 3' untranslated regions of the two mRNAs shared part of the last exon, which was 0.6 x 10(3) base-pairs long. There are two canonical acceptor sites available for RNA splicing in the last exon, the first being located at the 5' end of the exon, and the second at 370 base-pairs downstream from this end. Together with analysis by S1 nuclease mapping, the foregoing results lead us to conclude that, by the differential use of these two acceptor sites, a single gene generates two distinct mRNAs of 1.45 x 10(3) base-pairs and 1.1 x 10(3) base-pairs with or without the 5' half of the last exon. The two mRNAs appear to utilize the same modified poly(A) signal, AGTAAA, rather than the authentic AATAAA sequence present about 30 base-pairs downstream from the poly(A) attachment sites. This is probably because another consensus G + T-rich sequence is present at an appropriate distance from the AGTAAA sequence, but not from the AATAAA sequence. The gene for the cardiac myosin alkali light chain has proved to be expressed in ventricular muscle and in atrial and anterior latissimus dorsi muscles, the last of these being characteristic of slow skeletal muscle. In these muscles, two kinds of mRNA for the cardiac myosin alkali light chain, identical with those in ventricular muscle, were expressed and their relative amount in each tissue was almost the same as that in ventricular muscle.
Asunto(s)
Pollos/genética , Exones , Miosinas/genética , Empalme del ARN , Animales , Secuencia de Bases , Sitios de Unión , ADN Circular/genética , Genes , Datos de Secuencia Molecular , Músculos , Miocardio , Mapeo RestrictivoRESUMEN
A chick embryonic myosin alkali light chain L23 gene that is expressed transiently at embryonic stages in chick skeletal, cardiac and smooth muscles and in brain continuously from embryo to adult stages, was isolated and characterized. Sequence analysis showed that the exonic sequence of this gene was identical with that of embryonic myosin light chain mRNA except for one base replacement. This gene is a single gene of 5200 bases, which is divided into seven exons by six introns, and the positions of inserts of all the introns are well-conserved as in the skeletal and cardiac muscle myosin alkali light chain genes. Therefore, this embryonic myosin light chain gene can be classified as a member of the myosin alkali light chain gene family, and these three genes may have originated from a common ancestral gene. Transcription of the embryonic light chain gene starts from the same initiation site 33 bases upstream from ATG in embryonic muscle tissues and brain. Comparison of the nucleotide sequence around the promotor region of the embryonic myosin light chain gene with the corresponding regions of the skeletal and cardiac myosin light chain genes showed that the 11-base consensus sequence (TCCTATTTATAG) is present about 100 bases upstream from the transcription initiation site in each gene.
Asunto(s)
Proteínas Contráctiles/genética , Miosinas/genética , Fragmentos de Péptidos/genética , Animales , Secuencia de Bases , Embrión de Pollo , Regulación de la Expresión Génica , Genes , Molleja de las Aves/embriología , Datos de Secuencia Molecular , Músculos/análisis , Subfragmentos de Miosina , ARN Mensajero/genética , Mapeo Restrictivo , Transcripción GenéticaRESUMEN
Over 90% of congenital adrenal hyperplasia (CAH) results from 21-hydroxylase deficiency. Because the CYP21B gene is located within the HLA complex and is very tightly linked to HLA markers, HLA typing is widely used for prenatal diagnosis and identifying heterozygous family members. In the course of a study on identification of heterozygous family members with HLA typing, we recognized an unusual family case in which three siblings share the same HLA haplotype, and only one of them had the simple virilizing form; her two siblings did not have any endocrinological abnormalities. We investigated the mode of genetic transmission by using polymerase chain reaction and single stranded conformation polymorphism. The present study revealed that the proband was a compound heterozygote with the intron 2 mutation that causes aberrant RNA splicing and the missense mutation of exon 4, while the other siblings and the father had only one allele of a missense mutation in exon 4; the mother is a normal homozygote. This result together with DNA fingerprint analysis strongly suggest that the intron 2 mutation occurred de novo in the maternally inherited gene of the proband. This seems to be the first case of a de novo mutation of the CYP21B gene that causes CAH.
Asunto(s)
Hiperplasia Suprarrenal Congénita , Antígenos HLA/genética , Mutación , Esteroide 21-Hidroxilasa/genética , Hiperplasia Suprarrenal Congénita/enzimología , Hiperplasia Suprarrenal Congénita/genética , Secuencia de Bases , ADN/análisis , ADN/química , ADN/genética , Exones , Antígenos HLA/análisis , Haplotipos , Heterocigoto , Humanos , Intrones , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Linaje , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Empalme del ARNRESUMEN
A complete sequence of the Ah receptor gene was cloned from a mouse genomic library by using the Ah receptor cDNA as a probe. The Ah receptor gene is 37.5 kb long and is split into 11 exons by 10 introns. Sequence analysis of the 5' flanking region of the Ah receptor gene reveals that there is neither a TATA box nor a CAAT box in the promoter region. Instead, this gene has a few GC boxes and other enhancer elements in the 5' upstream flanking region. Southern blot analysis indicated that the Ah receptor gene is a unique gene.
Asunto(s)
Receptores de Hidrocarburo de Aril/genética , Animales , Secuencia de Bases , Clonación Molecular , ADN/genética , Cartilla de ADN/genética , ADN Complementario/genética , Elementos de Facilitación Genéticos , Exones , Biblioteca Genómica , Intrones , Ratones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , ARN Mensajero/genética , Mapeo Restrictivo , Transcripción GenéticaRESUMEN
Induction of cytochrome P4501A1 (CYP1A1) by certain xenobiotics is mediated by the Ah receptor/Arnt complex. The present knowledge about the molecular process of induction is summarized with special attention to our recent work on characterization of the polymorphic forms of the Ah receptor.
Asunto(s)
Sistema Enzimático del Citocromo P-450/biosíntesis , Sistema Enzimático del Citocromo P-450/genética , Polimorfismo Genético , Receptores de Hidrocarburo de Aril/genética , Xenobióticos/farmacología , Clonación Molecular , Inducción Enzimática/efectos de los fármacos , Humanos , Mutagénesis Sitio-Dirigida , Receptores de Hidrocarburo de Aril/química , Receptores de Hidrocarburo de Aril/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Xenobióticos/metabolismoRESUMEN
DNA synthesized by in vitro reverse transcription of the interferon mRNA has been cloned and amplified as recombinant DNA, TpIF319-13 (Taniguchi et al., 1979). The nucleotide sequence of this IF cDNA which consists of 770 bp (excluding the A:T tails) has been determined. The data reported predict the hitherto unknown amino acid sequence of human fibroblast interferon and its putative signal peptide.