RESUMEN
Mini-chromosome maintenance (Mcm) proteins are part of the replication-licensing complex that is loaded onto chromatin during the G1-phase of the cell cycle and required for initiation of DNA replication in the subsequent S-phase. Mcm proteins are typically loaded in excess of the number of locations that are used during S-phase. Nonetheless, partial depletion of Mcm proteins leads to cancers and stem cell deficiencies. Mcm2 deficient mice, on a 129Sv genetic background, display a high rate of thymic lymphoblastic lymphoma. Here array comparative genomic hybridization is used to characterize the genetic damage accruing in these tumors. The predominant events are deletions averaging less than 0.5 Mbp, considerably shorter than observed in prior studies using alternative mouse lymphoma models or human tumors. Such deletions facilitate identification of specific genes and pathways responsible for the tumors. Mutations in many genes that have been implicated in human lymphomas are recapitulated in this mouse model. These features, and the fact that the mutation underlying the accelerated genetic damage does not target a specific gene or pathway a priori, are valuable features of this mouse model for identification of tumor suppressor genes. Genes affected in all tumors include Pten, Tcfe2a, Mbd3 and Setd1b. Notch1 and additional genes are affected in subsets of tumors. The high frequency of relatively short deletions is consistent with elevated recombination between nearby stalled replication forks in Mcm2-deficient mice.
Asunto(s)
Proteínas de Ciclo Celular/deficiencia , Genes Relacionados con las Neoplasias , Proteínas Nucleares/deficiencia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Eliminación de Secuencia , Neoplasias del Timo/genética , Animales , Secuencia de Bases , Proteínas de Ciclo Celular/genética , Puntos de Rotura del Cromosoma , Cromosomas de los Mamíferos/genética , Hibridación Genómica Comparativa , Variaciones en el Número de Copia de ADN , Humanos , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Componente 2 del Complejo de Mantenimiento de Minicromosoma , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Análisis de Secuencia de ADNRESUMEN
Minichromosome maintenance proteins (Mcm's) are components of the DNA replication licensing complex. In vivo, reduced expression or activity of Mcm's has been shown to result in highly penetrant early onset cancers (Shima et al., 2007; Pruitt et al., 2007) and stem cell deficiencies (Pruitt et al., 2007). Here we use mouse embryonic fibroblasts from an Mcm2-deficient strain of mice to show by DNA fiber analysis that origin usage is decreased in Mcm2-deficient cells under conditions of hydroxyurea (HU)-mediated replication stress. DNA damage responses (DDRs) resulting from HU and additional replication-dependent and replication-independent genotoxic agents were also examined and shown to function at wild-type (wt) levels. Further, basal levels of many components of the DDR were expressed at wt levels, showing that there is no acute replicative stress under normal growth conditions. Only very modest, 1.5- to 2-fold increases in the basal levels of gamma-H2AX, p21(cip1) and 53bp foci were found, consistent with a slight chronic elevation in DDR pathways. The one condition in which a larger difference between wt- and Mcm2-deficient cells was found occurred after ultraviolet irradiation and may reflect the role of Chk1-mediated suppression of dormant origins. In vivo, abrogating p53-mediated DDR in Mcm2-deficient mice results in increased embryonic lethality and accelerated cancer formation in surviving mice. Further, p53 mutation rescues the negative effect of Mcm2 deficiency on the survival of neural stem cells in vitro; however, the enhanced survival correlates with increased genetic damage relative to Mcm2 wt cells carrying the p53 mutation. Together these results show that even relatively minor perturbations to primary or dormant replication origin usage contribute to accelerated genetic damage in vivo. In addition, these studies show that tumor types resulting from Mcm2 deficiency are strongly affected by interaction with both genetic background and p53.
Asunto(s)
Transformación Celular Neoplásica , Daño del ADN , Neoplasias/metabolismo , Neoplasias/patología , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Alelos , Animales , Línea Celular , Proliferación Celular , ADN/química , ADN/genética , Eliminación de Gen , Regulación de la Expresión Génica , Humanos , Hibridación Genética , Ratones , Componente 2 del Complejo de Mantenimiento de Minicromosoma , Neoplasias/genética , Sistema Nervioso/citología , Células Madre/citología , Células Madre/metabolismo , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Expression from the mouse Ren-1(c) gene in As4.1 cells is dependent on a proximal promoter element (PPE) located at approximately -60 and a 241-base pair enhancer region located at -2625 relative to the transcription start site. The PPE (TAATAAATCAA) is identical to a consensus HOX.PBX binding sequence. Further, PBX1b has been shown to be a component of a PPE-specific binding complex present in nuclear extracts from As4.1 cells. The binding affinities of different paralog HOX members to the PPE were examined in the absence or presence of PBX1b. HOXB6, -B7, and -C8 failed to bind the PPE alone but showed weak affinity in the presence of PBX1b. In contrast, HOXD10 and to a lesser degree HOXB9 bound the PPE with high affinities regardless of whether PBX1b was present. Abd-B HOX members, including HOXD10, -A10, -A9, -B9, and -C9, are expressed in As4.1 cells. The ability of HOX and PBX1b to form a ternary complex with PREP1 on the PPE is also demonstrated both in vivo and in vitro. Point mutations in either the HOX or PBX half-site of the PPE disrupted the formation of the HOX.PBX complex and dramatically decreased transcriptional activity of the Ren-1(c) gene demonstrating that both the HOX and PBX half-sites are critical for mouse renin gene expression. These results strongly implicate Abd-B class Hox genes and their cofactors as major determinants of the sites of renin expression.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación Enzimológica de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas/metabolismo , Renina/genética , Animales , Secuencia de Bases , Sitios de Unión , Línea Celular , Núcleo Celular/metabolismo , Secuencia de Consenso , Humanos , Ratones , Datos de Secuencia Molecular , Factor de Transcripción 1 de la Leucemia de Células Pre-B , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Homología de Secuencia de Ácido NucleicoRESUMEN
A reporter construct was assembled with 4-kb of renin 5'-flanking sequence fused to humanized green fluorescent protein (GFP) cDNA. Transgenic mice carrying this construct were produced and assayed for GFP expression. In the adult, expression was detected in juxtaglomerular (JG) cells of the kidney and granular convoluted tubular cells of the submandibular gland. Furthermore, treatment of mice with captopril induced GFP expression in renal vascular smooth muscle cells. During embryogenesis, GFP expression was first detected at embryonic day E13 in the adrenal gland and Wolffian duct. Expression was also seen in the developing renal vasculature as early as E14 and remained detectable through birth. Renal GFP expression became restricted to JG cells in adults. Fetal adrenal and gonadal arteries also expressed GFP. In the placenta, GFP was observed in giant cell trophoblasts, consistent with reports of renin expression in chorionic cells of both humans and mice. We conclude that 4 kb of renin 5' flank is sufficient to direct multiple known renin expression patterns. Furthermore, the renin-GFP construct characterized here will provide a useful vital reporter for renin expression.
Asunto(s)
Envejecimiento/genética , Embrión de Mamíferos/química , Embrión de Mamíferos/metabolismo , Regulación de la Expresión Génica , Proteínas Luminiscentes/genética , Renina/genética , Transgenes , Animales , Femenino , Técnicas de Transferencia de Gen , Proteínas Fluorescentes Verdes , Humanos , Riñón/irrigación sanguínea , Riñón/química , Riñón/metabolismo , Proteínas Luminiscentes/biosíntesis , Masculino , Ratones , Ratones Transgénicos , Placenta/química , Placenta/metabolismo , Embarazo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Renina/biosíntesis , Glándula Submandibular/química , Glándula Submandibular/metabolismo , Sistema Urogenital/química , Sistema Urogenital/metabolismoRESUMEN
The transcription start site and DNA sequence elements required for the induction of Pax3 expression in differentiating P19 embryonal carcinoma cells have been localized. These elements consist of a promoter and additional elements located within 1.6 kbp 5' to the transcription start site. Sequence elements within this 1.6 kbp region are also sufficient to mediate the induction and dorsal restriction of Pax3 in the neural tube and somites of transgenic mice throughout the hindbrain and trunk. Additional elements required for expression anterior to the hindbrain and in migrating myoblasts are located within 14 kbp 5' to the transcription start site. This region also contains element(s) that repress Pax3 expression in the ventral body wall mesoderm of the tail bud.
Asunto(s)
Proteínas de Unión al ADN/genética , Factores de Transcripción , Animales , Secuencia de Bases , Clonación Molecular , ADN , Regulación del Desarrollo de la Expresión Génica , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Factor de Transcripción PAX3 , Factores de Transcripción Paired Box , Regiones Promotoras Genéticas , Eliminación de Secuencia , Transcripción Genética , Células Tumorales CultivadasRESUMEN
A frameshift mutation is present in one allele of the Atf4 gene in genomic DNA from F9 embryonal carcinoma stem cells. The mutation results in the fusion of a short 5' open reading frame to the coding sequence of Atf4, replacing the first 18 N-terminal amino acids with 50 amino acids encoded by the upstream open reading frame. The ability of both normal and mutated Atf4 gene products to influence cell growth was tested using an NIH3T3 cell transformation assay. Overexpression of mutant Atf4 suppresses ras-induced transformation in this assay. In G418r cell lines derived from parallel co-transfections, expression of transfected mutant Atf4 mRNA correlates with a loss of transformed morphology and a reduction in ras mRNA levels. Transient cotransfection assays in NIH3T3 cells demonstrate that wild type Atf4 is able to inhibit transcription directed by the human c-Ha-ras1 promoter and that this effect is increased by the mutation.
Asunto(s)
Transformación Celular Neoplásica , Genes ras , Proteínas Proto-Oncogénicas p21(ras)/biosíntesis , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Células 3T3 , Factor de Transcripción Activador 4 , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Carcinoma Embrionario , Línea Celular , Cartilla de ADN , Mutación del Sistema de Lectura , Humanos , Cinética , Ratones , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Reacción en Cadena de la Polimerasa , ARN Mensajero/biosíntesis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
Endogenous signals capable of inducing neuroectodermal differentiation are expressed by differentiating P19 EC cells in vitro. The present study demonstrates that at least two discrete signals are required. One is expressed by isolated primitive streak mesoderm-like cell lines and has the capacity to induce the expression of Pax-3 but, alone, induces neural differentiation inefficiently. The second signal is not expressed by the primitive streak mesoderm-like cell line but is present in conditioned media from differentiating P19 EC cells following DMSO treatment. This signal does not induce either Pax-3 expression or morphological differentiation and does not commit stem cells to a neuroectodermal fate. Rather, it acts synergistically with the signal derived from the primitive streak mesoderm-like cells to increase the efficiency with which stem cells respond initially by Pax-3 expression and subsequently by differentiation towards neural lineages. The activity of this second signal can be replaced by forskolin and 3-isobutyl-1-methyl-xanthine suggesting that its effects are transduced by a cyclic nucleotide-dependent pathway.
Asunto(s)
Proteínas de Unión al ADN/genética , Inducción Embrionaria , Regulación de la Expresión Génica , Células Madre Neoplásicas/citología , Tejido Nervioso/citología , Transducción de Señal , Proteínas de Dominio T Box , Factores de Transcripción , 1-Metil-3-Isobutilxantina/farmacología , Animales , Northern Blotting , Colforsina/farmacología , Dimetilsulfóxido/farmacología , Células Madre de Carcinoma Embrionario , Proteínas Fetales/genética , Técnica del Anticuerpo Fluorescente , Expresión Génica , Histocitoquímica , Factor de Transcripción PAX3 , Factores de Transcripción Paired Box , Proteína Quinasa C/metabolismoRESUMEN
Differentiating P19 embryonal carcinoma (EC) cells transiently express an endogenous activity capable of inducing Pax-3 expression in adjacent P19 stem cells (Pruitt, Development 116, 573-583, 1992). In the present study, expression of this activity in mesodermal cell lineages is demonstrated. First, expression of the mesodermal marker Brachyury correlates with expression of Pax-3-inducing activity. Second, the ability of leukemia inhibitory factor (LIF) to block mesoderm differentiation at two different points is demonstrated and correlated with the inhibition of Pax-3-inducing activity. Finally, two mesodermal cell lines that express Pax-3-inducing activity were derived from P19 EC cells. Each of these lines expresses high levels of the mesodermal marker Brachyury and high levels of Oct-3/4 (which is down-regulated at early times during mesoderm differentiation) suggesting that these lines are early mesodermal derivatives. Unlike EC or embryonic stem cell lines, each of the two mesodermal derivatives autoinduces Hox gene expression on aggregation even in the presence of LIF. Following aggregation, anterior-specific genes are expressed more rapidly than more posterior genes. These observations directly demonstrate the ability of murine mesodermal derivatives to autoinduce Hox gene expression in the absence of signals from other cell lineages. Similar to the Pax-3-inducing activity, signals from mesodermal cell lines were sufficient to induce HOX expression in adjacent P19 stem cells in cell mixing assays. These observations are consistent with the previous suggestion (Blum, M., Gaunt, S. J., Cho, K. W. Y., Steinbeisser, H., Blumberg, B., Bittner, D. and De Robertis, E. M. (1992) Cell 69, 1097-1106) that signals responsible for anterior-posterior organizer activity are localized to the anterior primitive streak mesoderm of the mouse embryo.
Asunto(s)
Proteínas de Unión al ADN/genética , Inducción Embrionaria/genética , Gástrula/fisiología , Regulación de la Expresión Génica/genética , Genes Homeobox/genética , Interleucina-6 , Mesodermo/fisiología , Factores de Transcripción , Proteínas de Xenopus , Animales , Northern Blotting , Southern Blotting , Línea Celular , Técnica del Anticuerpo Fluorescente , Marcadores Genéticos , Técnicas Genéticas , Inhibidores de Crecimiento/fisiología , Factor Inhibidor de Leucemia , Linfocinas/fisiología , Ratones , Factor de Transcripción PAX3 , Factores de Transcripción Paired Box , Células Tumorales Cultivadas , XenopusRESUMEN
Restriction fragment length variant analysis employing a mouse cDNA probe was used to localize the gene encoding murine activating transcription factor 4 (ATF-4) to mouse chromosome 15 in close proximity to Sis (the cellular homolog of the simian sarcoma virus oncoprotein). Previous studies suggest that conserved linkage relationships exist between this region of mouse chromosome 15 and human chromosome 22q. The chromosomal locations of genes encoding most members of the ATF and cyclic AMP response element binding protein (CREB) subfamily of b-zip proteins have not been determined. This study demonstrates that the location of the gene for murine ATF-4 is not linked to the genes for JUN family members, CREB1 and CREB2. Further mapping of individual ATF/CREB subfamily members in the mouse will provide insight into the evolution of this multigene family.
Asunto(s)
Mapeo Cromosómico , Factores de Transcripción/genética , Factor de Transcripción Activador 4 , Animales , Cruzamientos Genéticos , ADN , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBARESUMEN
A P19 embryonal carcinoma stem cell line carrying an insertion of the E. coli LacZ gene in an endogenous copy of the Pax-3 gene was identified. Expression of the Pax-3/LacZ fusion gene in neuroectodermal and mesodermal lineages following induction of differentiation by chemical treatments (retinoic acid and dimethylsulfoxide) was characterized using this line and is consistent with the previous localization of Pax-3 expression in the embryo to mitotically active cells of the dorsal neuroectoderm and the adjacent segmented dermomyotome. Pax-3/LacZ marked stem cells were also utilized as target cells in mixing experiments with unmarked P19 cells that had been differentiated by pretreatment with chemical inducers. Induction of beta-galactosidase and neuroectodermal markers in the target cells demonstrates that: (1) some differentiated P19 cell derivatives transiently express endogenous Pax-3- and neuroectoderm-inducing activities, (2) undifferentiated target stem cells respond to these activities even in the presence of leukemia inhibitory factor and (3) the endogenous activities can be distinguished from, and are more potent than, retinoic acid treatment in inducing neuroectoderm. These observations demonstrate that P19 embryonal carcinoma cells provide a useful in vitro system for analysis of the cellular interactions responsible for neuroectoderm induction in mammals.
Asunto(s)
Proteínas de Unión al ADN/genética , Ectodermo/fisiología , Inducción Embrionaria/fisiología , Expresión Génica/fisiología , Mamíferos/embriología , Sistema Nervioso/embriología , Animales , Northern Blotting , Línea Celular , Dimetilsulfóxido/farmacología , Inducción Embrionaria/efectos de los fármacos , Técnica del Anticuerpo Fluorescente , Mesodermo/fisiología , Teratoma , Tretinoina/farmacología , Células Tumorales CultivadasRESUMEN
The ability of leukemia inhibitory factor (LIF) to block differentiation of P19 embryonal carcinoma (EC) cells under a variety of induction conditions was determined. LIF inhibits differentiation under several conditions which lead to endodermal and mesodermal cell lineages including skeletal and cardiac muscle. In contrast, LIF does not block differentiation when cells are induced under conditions which lead to neuro-ectodermal cell types including neurons and astroglial cells. These studies demonstrate that P19 EC cell differentiation can be divided into LIF sensitive and insensitive pathways which correlate with differentiation of endodermal/mesodermal and neuro-ectodermal cell types, respectively. The effect of LIF on mRNA levels for several genes which have previously been implicated in mediating differentiation in P19 EC cells was determined. LIF has no effect on the mRNA levels for retinoic acid receptor (RAR) alpha, RAR beta, RAR gamma, jun A, jun D, c-fos, or fra-1. In contrast LIF stimulates jun B mRNA expression by a factor of four to six under all induction conditions.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Inhibidores de Crecimiento/farmacología , Interleucina-6 , Linfocinas/farmacología , ARN Mensajero/metabolismo , Tretinoina/farmacología , Carcinoma/embriología , Proteínas Portadoras/análisis , Humanos , Factor Inhibidor de Leucemia , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , Receptores de Ácido Retinoico , Células Tumorales CultivadasRESUMEN
Independent of de novo protein synthesis, interleukin-1, interleukin-6, and dexamethasone caused immediate stimulation of transcriptional activity of most major acute phase plasma protein genes in the rat hepatoma H-35 cells. However, activation of alpha 2-macroglobulin and alpha 1-acid glycoprotein genes were delayed by 2-4 h and required ongoing protein synthesis. The hormones also increased transiently the transcription of the junB gene and the amounts of JunB, C/EBP, and C/EBP-like mRNA. To identify whether JunB and C/EBP have the ability to control both the early and late acute phase reactants, expression vectors for mouse C/EBP and JunB together with reporter gene constructs containing recognized hormone-specific regulatory elements were introduced into hepatoma cells. C/EBP displayed prominent transactivation activity with the interleukin-1 and glucocorticoid regulatory elements of alpha 1-acid glycoprotein, the interleukin-1 regulatory element of haptoglobin gene, and the interleukin-6 regulatory element of beta-fibrinogen. The interleukin-6 regulatory elements of the first two genes and the glucocorticoid response element of the third gene were not affected by C/EBP. These data suggest that normal hormone activation of these three acute phase reactant genes might involve, in part, C/EBP-related factors which have a broad range of specificity. H-35 cells stably transformed with a mouse C/EBP expression vector showed an elevated basal level as well as cytokine inducible expression of some but not all acute phase reactants. Cotransfected JunB resulted in reduced activity of cytokine-responsive constructs and in lower transactivation by C/EBP. JunB appears to function as a modulator of plasma protein expression during the course of acute phase response.
Asunto(s)
Proteínas de Fase Aguda/genética , Proteínas Sanguíneas/genética , Citocinas/metabolismo , Glucocorticoides/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción , Transcripción Genética , Animales , Proteínas Potenciadoras de Unión a CCAAT , Línea Celular Transformada , Cloranfenicol O-Acetiltransferasa/genética , Cicloheximida/farmacología , Proteínas de Unión al ADN , Regulación de la Expresión Génica , Plásmidos , Ratas , TransfecciónRESUMEN
A set of vectors is described which allow the efficient cloning of full-length cDNAs, using a modification of the method of Okayama and Berg [Mol. Cell Biol. 2 (1982) 161-170], and enrichment of specific sequences directly from cDNA libraries by hybridization/selection. The vectors pcDpolyB+ and pcDpolyB- are derived from an expression vector described previously [Okayama and Berg, Mol. Cell Biol. 3 (1983) 280-289] and allow expression of cloned cDNAs in eukaryotic cells from the simian virus 40 early region promoter. The vectors BSB+ and BSB- contain convenient priming sites for sequence analysis and the T3 and T7 RNA polymerase promoters, allowing synthesis of transcripts homologous to either strand of the cDNA. Each of these vectors also contains the intergenic region from the bacteriophage f1 permitting synthesis of single-stranded (ss) copies of the cDNA libraries. Enrichment for cDNAs containing sequences homologous to the hypoxanthine phosphoribosyl transferase gene from an ss copy of a cDNA library by hybridization/selection is demonstrated. Levels of enrichment sufficient for the direct cloning of specific sequences without requiring colony or plaque hybridizations were obtained. Libraries constructed from different cell types can be screened against each other to create sublibraries highly enriched in sequences specific to a single cell type. The availability of cDNA expression libraries enriched for cell-type-specific cDNAs should greatly enhance the efficiency with which cDNAs can be identified on the basis of functional assays.
Asunto(s)
Clonación Molecular/métodos , ADN , Desoxirribonucleasas de Localización Especificada Tipo II , Vectores Genéticos , Hibridación de Ácido Nucleico , Plásmidos , Enzimas de Restricción del ADN , ADN Circular/genética , ADN de Cadena Simple/genética , ADN de Cadena Simple/aislamiento & purificación , Eficiencia , Escherichia coli/genética , Hipoxantina Fosforribosiltransferasa/genética , Oligodesoxirribonucleótidos/genética , Transformación GenéticaRESUMEN
We have analyzed the effect of DNA intercalating agents on the transcription signals from two different Xenopus laevis RNA polymerase I promoters. The transcription signal from the promoter for the 7.5-kilobase rRNA precursor (the gene promoter) is unaffected over a large range of intercalating agent concentrations regardless of whether the template is injected plasmid DNA in oocytes, the amplified endogenous nucleoli of oocytes, or the endogenous chromosomes of cultured Xenopus kidney cells. The transcription signal from a closely related promoter located in the spacer DNA between genes (the spacer promoter) ranges between undetectable to equivalent to the gene promoter signal on different templates. The transcription signal from the spacer promoter is also differentially affected by intercalating agents relative to the gene promoter. Depending on the template, these agents can either increase or decrease the transcription signal from the spacer promoter. Fusions between the gene and spacer promoters demonstrate that intercalating agents affect transcription initiation. One explanation for these results is that the degree of supercoiling of the template DNA can differentially inhibit transcription from the spacer promoters. The different effects of intercalating agents on transcription from the spacer promoters of various templates could then be explained as differences in the degree of supercoiling present on these templates initially.
Asunto(s)
Sustancias Intercalantes/farmacología , Operón , ARN Polimerasa I/genética , Animales , Secuencia de Bases , Femenino , Conformación de Ácido Nucleico , Oocitos , Plásmidos , Regiones Promotoras Genéticas , Transcripción Genética , Xenopus laevisRESUMEN
We have analyzed the effects of topological constraint on the transcription of both injected ribosomal DNA plasmids and the endogenous ribosomal genes in Xenopus oocytes. Efficient transcription of injected ribosomal gene plasmids requires a covalently closed circular template. Once transcription is initiated on injected plasmids there is a continuous requirement for topological constraint, since subsequent cutting with a restriction endonuclease abolishes transcription. In contrast, both initiation and elongation of transcription on endogenous ribosomal genes are maintained after cutting with restriction endonucleases.
Asunto(s)
ADN/genética , Genes , Oocitos/metabolismo , Plásmidos , Transcripción Genética , Animales , Enzimas de Restricción del ADN , ADN Circular/genética , ADN Ribosómico , Electroforesis en Gel de Poliacrilamida , Femenino , Microscopía Electrónica , Conformación de Ácido Nucleico , XenopusRESUMEN
We report an analysis by electron microscopy of the differences in the folding of ribosomal gene and adjacent nontranscribed spacer DNA of Xenopus laevis oocytes into supranucleosomal chromatin structures. The chromatin structures identified in gene and spacer regions of transcriptionally active nucleoli (from stage 5 oocytes) were compared with those found in nucleoli prior to transcription (from stage 2 oocytes) to determine whether changes in the chromatin structure occur when transcription is initiated. Chromatin structures were characterized by their morphology and by the extent of folding of DNA in chromatin. Nontranscribed spacer regions from both transcriptionally active and inactive nucleoli appear to be packaged into supranucleosomal structures and are contracted by a factor of at least 20 from the length of B form DNA. The structure of the adjacent gene region, both before and during transcription, is much more extended; the only structures observed are the size of nucleosomes, and the DNA is contracted by a factor of 1.4 from its B form length. Thus a mosaicism in the higher order structure of gene and spacer rDNA is established days or weeks prior to the initiation of transcription of these genes and maintained during transcriptionally active stages.
Asunto(s)
Nucléolo Celular/ultraestructura , Cromatina/ultraestructura , ARN Ribosómico/genética , Transcripción Genética , Animales , ADN Recombinante , ADN Ribosómico , Microscopía Electrónica , Nucleosomas/ultraestructura , Xenopus laevisRESUMEN
The organization of nucleosomes in higher order chromatin structures has been studied by electron microscopy of chick red blood cell nuclei. Chromatin appears as a thick fiber with an average diameter of approximately 300 A when prepared for electron microscopy in buffers which approximate physiological ionic strength. Progressive steps of disassembly of the thick fiber into individual nucleosomes could be induced either by ionic strength reduction or by tRNA treatment (which removes histone H1 and some non-histone chromosomal proteins). When disassembly was induced by ionic strength reduction in the presence of Mg++ (or Ca++), the lengths of the intermediate disassembly products were found to be multiples of 330 A. The diameter of these structures was estimated to be 275 A. This intermediate in the disassembly process is not observed if thick fiber disassembly is induced by ionic strength reduction in the absence of divalent cations. To investigate whether the higher order structural unit is present in the thick fiber at physiological ionic strengths, tRNA treatment was used to induce thick fiber disassembly under physiological monovalent ionic conditions. In this case, either with or without divalent cations, a supranucleosomal unit was found with dimensions similar to those given above. This data provides evidence for a slightly oblong supranucleosomal structure (330 x 275 A) whick forms a repeating unit in the chromatin thick fiber.