RESUMEN
Synthesis of H1° histone, in the developing rat brain, is also regulated at post-transcriptional level. Regulation of RNA metabolism depends on a series of RNA-binding proteins (RBPs); therefore, we searched for H1° mRNA-interacting proteins. With this aim, we used in vitro transcribed, biotinylated H1° RNA as bait to isolate, by a chromatographic approach, proteins which interact with this mRNA, in the nuclei of brain cells. Abundant RBPs, such as heterogeneous nuclear ribonucleoprotein (hnRNP) K and hnRNP A1, and molecular chaperones (heat shock cognate 70, Hsc70) were identified by mass spectrometry. Western blot analysis also revealed the presence of cold shock domain-containing protein 2 (CSD-C2, also known as PIPPin), a brain-enriched RBP previously described in our laboratory. Co-immunoprecipitation assays were performed to investigate the possibility that identified proteins interact with each other and with other nuclear proteins. We found that hnRNP K interacts with both hnRNP A1 and Hsc70 whereas there is no interaction between hnRNP A1 and Hsc70. Moreover, CSD-C2 interacts with hnRNP A1, Y box-binding protein 1 (YB-1), and hnRNP K. We also have indications that CSD-C2 interacts with Hsc70. Overall, we have contributed to the molecular characterization of a ribonucleoprotein particle possibly controlling H1° histone expression in the brain.
Asunto(s)
Encéfalo/metabolismo , Proteínas del Choque Térmico HSC70/metabolismo , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Histonas/metabolismo , ARN Mensajero/metabolismo , Animales , Proteínas del Choque Térmico HSC70/genética , Ribonucleoproteínas Nucleares Heterogéneas/genética , Histonas/genética , Espectrometría de Masas , ARN Mensajero/genética , Ratas , Ratas WistarRESUMEN
Cytochrome c oxidase (COX) complex is built up with both nucleus- and mitochondrion-encoded subunits. Biogenesis and assembly of the complex thus requires fine cross-talk between the two compartments. In order to shed light on the regulation of nuclear-mitochondrial interactions, we studied the expression of COXIII (mitochondrion-encoded) and COXIV (nucleus-encoded) in adult rat tissues and rat developing brain. We found that the levels of COXIV protein and mRNA are not linearly related, thus suggesting a post-transcriptional mode of regulation. In agreement with this observation, we report the presence of a protein that specifically binds to the 3'-untranslated region of COXIV mRNA. This factor, that forms with RNA a complex of about 60 kDa, is present both in the cytoplasm and mitochondria, where its concentration decreases throughout development with inverse correlation with COXIV accumulation. Interestingly, using an antibody raised in our laboratory, we found that, in developing rat brain, COXIII does not localize exclusively to mitochondria, but is also present in the cytosol, where it could exert a yet unknown regulatory role.
Asunto(s)
Encéfalo/embriología , Encéfalo/metabolismo , Complejo IV de Transporte de Electrones/biosíntesis , Procesamiento Postranscripcional del ARN/fisiología , Animales , Northern Blotting , Western Blotting , Citoplasma/metabolismo , Electroforesis en Gel Bidimensional , Regulación Enzimológica de la Expresión Génica , Mitocondrias/metabolismo , Transporte de Proteínas/fisiología , Proteínas/análisis , ARN Mensajero/análisis , RatasRESUMEN
Drug delivery to the central nervous system (CNS) is subject to the permeability limitations imposed by the blood-brain barrier (BBB). Several systems in vitro have been described to reproduce the physical and biochemical behavior of intact BBB, most of which lack the feature of the in vivo barrier. We developed a fully formed monolayer of RBE4.B immortalized rat brain microvessel endothelial cells (ECs), grown on top of polycarbonate filter inserts with cortical neuronal cells grown on the outside. Neurons induce ECs to synthesize and sort occludin to the cell periphery. Occludin localization is regulated by both compositions of the substratum and soluble signals released by cortical co-cultured neurons. The observed effects do not require strict physical contact among cells and neurons. To assess the physiological function of the barrier we examined the transendothelial transfer of three test compounds: dopamine, L-tryptophan and L-DOPA. Polycarbonate filter inserts, where ECs were co-cultured with neurons, were assumed as open two compartments vertical dynamic models. Permeation studies demonstrated that the ECs/neurons co-cultures possess permeability characteristics approaching those of a functional BBB: the system behaved as a selective interface that excludes dopamine permeation, yet permits L-tryptophan and L-DOPA to cross. The movement of test compounds from the donor to the acceptor compartment was observed at a distinct time from the start of co-culture. Transfer was determined using standard kinetic equations. Different performance was observed after 5 and 7 days of co-culture. After 5 days dopamine, L-tryptophan and L-DOPA passively permeate through the membrane as indicated by fittings with a first-order kinetic process equation. After 7 days of co-culture, occludin localizes at ECs periphery, dopamine does not cross the barrier to any further extent, while the transfer of L-tryptophan and L-DOPA fits well with a saturable Michaelis-Menten kinetic process, thus indicating the involvement of a specific carrier-mediated transport mechanism. Permeation studies confirmed that culture of ECs in the presence of neurons induces the characteristic permeability limitations of a functional BBB.
Asunto(s)
Barrera Hematoencefálica , Animales , Células Cultivadas , Dopamina/farmacocinética , Levodopa/farmacocinética , Permeabilidad , Ratas , Ratas Sprague-Dawley , Triptófano/farmacocinéticaRESUMEN
During aging rat myocardium undergoes structural changes characterized by a shift in the synthesis of myosin heavy chain (MHC) from V1 isoform, composed of two alpha-MHC, to V3 isoform, composed of two beta-MHC. In rat, besides ageing, cardiac hypertrophy as adaptive response to a superimposed pressure load (such as hypertension) is characterized by predominance of V3 myosin isoform. The aim of our study was to evaluate the expression of beta-MHC in right (RV) and left (LV) ventricles of spontaneously hypertensive rats (SHRs), a well defined animal model of hypertension, in relation to aging. We used very young (8-week old) and young (15-week old) SHRs and age-matched normotensive Harlan Sprague-Dawley control rats. By Western analysis, we found that beta-MHC is already present in both RV and LV of 8-week old SHRs, and is markedly predominant in RV and LV of 15-week old SHRs, when compared with age-matched control rats. Our study showed that the shift to V3 myosin isoform in SHRs is an early event, resembling accelerated senescence. We have also demonstrated that beta-MHC is actively synthesized also in young (15-week old) normal rats.
Asunto(s)
Envejecimiento/fisiología , Ventrículos Cardíacos/metabolismo , Hipertensión/fisiopatología , Cadenas Pesadas de Miosina/metabolismo , Animales , Western Blotting , Masculino , Isoformas de Proteínas/metabolismo , Ratas , Ratas Endogámicas SHRRESUMEN
Thyroid hormones (THs) enhance MHC alpha gene- and repress MHC beta gene-transcription in the heart, by interacting with specific nuclear receptors (TRs), that bind to regulatory sequences localized upstream of basal promoter of myosin heavy chain (MHC) genes. The overall effects of THs include an increase in V1- and a decrease in V3-myosin isozyme concentration in the heart. Myosin V1 contains two MHC alpha chains and has a higher ATPase activity than V3 isoform, which contains two beta chains. Previous studies on papillary muscles of spontaneously hypertensive rats (SHRs) showed that heart hypertrophy is accompanied by a shift from alpha to beta MHC accumulation. The present study was aimed at evaluating whether this event relates to differential expression of alpha1, alpha2, and beta1 isoforms of TRs. At the ages of 8 and 15 weeks, SHRs and Harlan Sprague-Dawley control rats were sacrificed under anesthesia and their hearts were dissected into left and right ventricles, free of atria and great vessels. The results of Western blot analyses showed that the levels of the three TR isoforms do not differ significantly between SHRs and control rats of the same age, either in the left or in the right ventricle. Thus, the expression of MHC beta in SHR hypertrophic heart does not seem to depend on changes in TR isoform concentrations.
Asunto(s)
Cardiomegalia/metabolismo , Hipertensión/complicaciones , Isoformas de Proteínas/metabolismo , Receptores de Hormona Tiroidea/metabolismo , Animales , Western Blotting , Cardiomegalia/etiología , Ventrículos Cardíacos/química , Masculino , Modelos Animales , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Ratas , Ratas Endogámicas SHR , Ratas Sprague-DawleyRESUMEN
We recently cloned a cDNA encoding an RNA-binding protein, that we called PIPPin, which is highly enriched in the rat brain and contains two putative double stranded RNA-binding domains (PIP1 and PIP2) and a central cold shock domain (CSD). Here we report that PIPPin is specifically enriched in some pyramidal neurons of the cerebral cortex and in the Purkinje cells of the cerebellum. We also show that PIPPin inhibits translation of H1(o) and H3.3 mRNA in a cell-free system. The results reported suggest that PIPPin down-regulates histone variant expression in the developing rat brain.
Asunto(s)
Cerebelo/metabolismo , Corteza Cerebral/metabolismo , Regulación de la Expresión Génica , Histonas/genética , Proteínas del Tejido Nervioso/metabolismo , Células de Purkinje/metabolismo , Células Piramidales/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Proteínas del Tejido Nervioso/análisis , Biosíntesis de Proteínas , ARN Mensajero/genética , Proteínas de Unión al ARN/análisis , Conejos , Ratas , Ratas Sprague-DawleyRESUMEN
We report that extracellular matrix and neurons modulate the expression of occludin, one of the main components of tight junctions, by rat brain endothelial cells (RBE4.B). Of the three extracellular matrix proteins which we tested (collagen I, collagen IV, and laminin), collagen IV stimulated at the best the expression of occludin mRNA. The corresponding protein, however, was not synthesized. Significant amounts of occludin accumulated only when RBE4.B cells were cultured on collagen IV-coated inserts, in the presence of cortical neurons, plated on laminin-coated companion wells. Finally, occludin segregated at the cell periphery, only when endothelial cells were co-cultured with neurons for > or = 1 week.
Asunto(s)
Encéfalo/metabolismo , Endotelio Vascular/química , Endotelio Vascular/metabolismo , Matriz Extracelular/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Neuronas/metabolismo , Animales , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/fisiología , Encéfalo/citología , Células Cultivadas , Circulación Cerebrovascular/fisiología , Colágeno/farmacología , Endotelio Vascular/efectos de los fármacos , Matriz Extracelular/efectos de los fármacos , Feto , Proteína Ácida Fibrilar de la Glía/metabolismo , Inmunohistoquímica , Laminina/farmacología , Proteínas de Neurofilamentos/metabolismo , Neuronas/efectos de los fármacos , Ocludina , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/metabolismo , Factores de TiempoRESUMEN
Growing evidence demonstrates the importance of regulating mRNA localization, stability and translation, in the control of gene expression, both in development and in differentiated cells. The signals responsible for specific regulation of mRNA metabolism reside in the RNA message itself: both 5' and 3' to the coding region, all transcripts contain variable lengths of untranslated sequences (5'-UTR and 3'-UTR) which contain the binding sites for a number of RNA-binding proteins (RBPs). Most RBPs assemble on the message at the moment of transcription, thus determining the future fate of the transcript from the very beginning. We discuss possible mechanisms through which mRNA, leaving from the nucleus as an RNA-protein complex, might reach its final intracellular destinations and how its access to the translational apparatus might be regulated in time and space. We also focus on a few known examples of aberrant RNA-protein interactions associated with human diseases, including cancer.
Asunto(s)
Proteínas/metabolismo , Estabilidad del ARN , ARN Mensajero/metabolismo , Regiones no Traducidas 3' , Regiones no Traducidas 5' , Secuencia de Aminoácidos , Animales , Núcleo Celular/metabolismo , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Biosíntesis de ProteínasRESUMEN
Chaperonins are ubiquitous proteins that facilitate protein folding in an adenosine triphosphate-dependent manner. Here we report the isolation of a sea urchin cDNA (Plhsp60) coding for mitochondrial chaperonin (Cpn60), whose basal expression is further enhanced by heat shock. The described cDNA corresponds to a full-length mRNA encoding a protein of 582 amino acids, the first 32 of which constitute a putative mitochondrial targeting leader sequence. Comparative analysis has demonstrated that this protein is highly conserved in evolution.
Asunto(s)
Chaperonina 60/genética , Erizos de Mar/genética , Secuencia de Aminoácidos , Animales , Chaperonina 60/metabolismo , ADN Complementario/genética , Embrión no Mamífero/metabolismo , Respuesta al Choque Térmico , Mitocondrias/genética , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Erizos de Mar/metabolismoRESUMEN
During maturation of mammalian brain, variants of both linker (i.e. H1 degrees) and core (i.e. H3.3) histone proteins accumulate in nerve cells. As the concentration of the corresponding transcripts decreases, in postmitotic cells, even if the genes are actively transcribed, it is likely that regulation of variant histone expression has relevant post-transcriptional components and that cellular factors affect histone mRNA stability and/or translation. Here we report that PIPPin, a protein that is highly enriched in the rat brain and contains a cold-shock domain, binds with high specificity to the transcripts that encode H1 degrees and H3.3 histone variants. Both mRNAs are bound through the very end of their 3'-untranslated region that encompasses the polyadenylation signal. Although PIPPin is present both in the cytoplasm and the nucleus of nerve cells, PIPPin-RNA complexes can be obtained only from nuclear extracts. The results of two-dimensional electrophoretic analysis suggest that a relevant proportion of nuclear PIPPin is more acidic than expected, thus suggesting that its RNA binding activity might be modulated by post-translational modifications, such as phosphorylation.
Asunto(s)
Química Encefálica , Histonas/genética , Proteínas del Tejido Nervioso/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Regiones no Traducidas 3' , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Núcleo Celular/química , Células Cultivadas , Frío , Femenino , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/análisis , Proteínas de Unión al ARN/análisis , Conejos , Ratas , Ratas Sprague-DawleyRESUMEN
This study is aimed to elucidate whether triiodothyronine (T3) and laminin have additive effects in regulating neural differentiation. We focused our attention on the expression of synapsin I, the 68 kDa component of the neurofilament triplet (NF-68), growth associated protein (GAP)-43 and microtubule associated protein (MAP)-2 as markers of synapses, cytoskeleton, axons and the somatodendritic domain, respectively. The addition of T3 to the medium of differentiating rat cortical neurons cultured on laminin did not have any effect on the concentration of these proteins, but was critical for their subcellular localization, suggesting a synergistic role of thyroid hormones and laminin in the establishment of neural polarity.
Asunto(s)
Polaridad Celular/efectos de los fármacos , Corteza Cerebral/citología , Laminina/farmacología , Proteínas del Tejido Nervioso/análisis , Neuronas/efectos de los fármacos , Triyodotironina/farmacología , Animales , Células Cultivadas , Corteza Cerebral/fisiología , Feto , Proteína GAP-43/análisis , Proteínas Asociadas a Microtúbulos/análisis , Proteínas de Neurofilamentos/análisis , Neuronas/citología , Neuronas/fisiología , Ratas , Ratas Sprague-Dawley , Sinapsinas/análisisRESUMEN
During brain maturation, histone H1(0) accumulates in both nerve and glial cells. The expression of this "linker" histone, the role of which still remains unclear, is a complex process, having both transcriptional and post-transcriptional regulatory components. In particular, the expression of H1(0) in rat cortical neurons is regulated mainly at the post-transcriptional level, and unknown cellular proteins are likely to affect H1(0) mRNA stability and/or translation. In looking for such factors, we tested the ability of rat brain extracts to protect H1(0) RNA probe from degradation by T1 RNase. The results reported here demonstrate that rat brain contains at least one major (p40) and two minor (p110 and p70) binding factors, specific for H1(0) RNA, all of which are much more or exclusively expressed in adult rat brain, when compared with other tissues. The binding of the factors is confined to a portion of the 3'-untranslated region (3'-UTR), which is highly conserved among murine and human H1(0) mRNAs. These findings suggest that the proteins identified play a critical role in regulating the expression of H1(0) histone in the brain of mammals.
Asunto(s)
Encéfalo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Histonas/genética , Proteínas de Unión al ARN/genética , Animales , Secuencia de Bases , Cartilla de ADN , Histonas/metabolismo , Datos de Secuencia Molecular , Unión Proteica , Sondas ARN , Proteínas de Unión al ARN/metabolismo , RatasRESUMEN
1. Neuronal differentiation depends on crosstalk between genetic program and environmental cues. In this study we tried to dissect this complex interplay by culturing neurons from fetal rat brain cortices in a chemically defined, neuron-specific, medium and on different substrata, either artificial (poly-D-lysine) or natural. 2. Among the extracellular matrix compounds used in this study, two (collagen I and fibronectin) allowed only a weak attachment of cortical neurons to the substratum, while the others (collagen IV, laminin, and basal lamina from Engelbreth-Holm-Swarm sarcoma) allowed both firm attachment and moderate to extensive neurite outgrowth from neuronal cell bodies. 3. By using synapsin I gene expression as a parameter of neuronal differentiation, we found that neurite outgrowth and neuronal differentiation are not linearly linked. Synapsin I gene expression, in fact, was maximal in neurons cultured on laminin, while the fastest neuritic outgrowth was recorded in cultures on poly-D-lysine. 4. The data presented in this paper are consistent with the hypothesis that the extracellular matrix plays an active role in modulating the differentiative program of neurons.
Asunto(s)
Corteza Cerebral/fisiología , Matriz Extracelular/fisiología , Regulación de la Expresión Génica/fisiología , Neuronas/fisiología , Sinapsinas/biosíntesis , Animales , Adhesión Celular , Diferenciación Celular , Células Cultivadas , Colágeno/farmacología , Combinación de Medicamentos , Feto , Fibronectinas/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Laminina/farmacología , Ratones , Neuritas/fisiología , Neuronas/citología , Neuronas/efectos de los fármacos , Polilisina/farmacología , Proteoglicanos , Ratas , Ratas Sprague-Dawley , Sarcoma Experimental , Células Tumorales CultivadasRESUMEN
The interactions of the iron complexes of the anthracycline antitumour drugs daunomycin (DN) and adriamycin (ADM) with the mononucleotide AMP, herring sperm DNA, plasmic pBR322 and immortalized 3T3 fibroblasts were studied. By means of Mössbauer spectroscopy it was demonstrated that DNA is a powerful ferric iron chelator as compared with AMP, which is not able to compete with DN or acetohydroxamic acid for ferric iron. The difference between AMP and DNA is postulated to be based on the chelate effect. The Mössbauer spectra of the ternary Fe-anthracycline-DNA systems differ from Fe-anthracycline binary complexes, indicating rearrangement reactions. Dialysis experiments clearly disclose the formation of a ternary Fe-ADM-pBR322 complex, the topology of which differs substantially from intercalating ADM. The effect of Fe-ADM complexes (3:1) on the growth of immortalized mouse embryonal fibroblasts (NIH-3T3) was studied in comparison with ADM alone. No significant difference on the inhibition of cell growth was noticed, suggesting comparable cytotoxicity for the compounds. In contrast to literature data, no evidence was found for DNA cleavage by ferric ADM at molar ratios as high as 1/100 (ADM/base pair), even if the ternary systems were prepared in the light and in the presence of reducing or oxidizing agents. Based on our observations it seems that the cytotoxicity of both ADM and Fe-ADM oligomer is not based primarily on intercalation or direct interaction with DNA.
Asunto(s)
Adenosina Monofosfato/química , División Celular/efectos de los fármacos , ADN/química , Daunorrubicina/química , Doxorrubicina/química , Hierro/química , Células 3T3 , Adenosina Monofosfato/farmacología , Animales , Transporte Biológico , ADN/farmacología , Daunorrubicina/metabolismo , Daunorrubicina/toxicidad , Doxorrubicina/metabolismo , Doxorrubicina/toxicidad , Peces , Hierro/toxicidad , Masculino , Ratones , Plásmidos/química , Espectroscopía de Mossbauer , EspermatozoidesRESUMEN
In looking for genes encoding RNA-binding factors, we prepared an expression library in lambda gt11, by cloning cDNAs corresponding to the polyadenylated fraction of RNA from rat brain at the embryonal day 18. The library was then screened by binding to a radioactive RNA, transcribed in vitro from a cDNA encoding the rat histone variant H3.3. Here we report some findings concerning a cDNA for a protein which contains two putative double stranded RNA-binding domains (dsBD). The corresponding message is specifically expressed in the brain. Moreover, Southern blot analysis showed that the gene is highly conserved from Drosophila melanogaster to man.
Asunto(s)
Encéfalo/metabolismo , Expresión Génica , Proteínas del Tejido Nervioso/biosíntesis , Proteínas de Unión al ARN/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Secuencia de Consenso , Secuencia Conservada , ADN Complementario , Drosophila melanogaster , Biblioteca de Genes , Humanos , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/química , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Proteínas de Unión al ARN/química , Ratas , Mapeo Restrictivo , Homología de Secuencia de AminoácidoRESUMEN
Accumulation of mRNAs encoding H1 zero and H3.3, two histone replacement variants, was studied in differentiating cortical neurons, cultured in a serum-free medium, with or without triiodothyronine (T3) supplementation. We found that the levels of both H1 (zero) and H3.3B mRNAs decrease in isolated neurons between the 2nd and 5th day of culture to the same extent as in vivo. At the same time, an active synthesis of the corresponding proteins was evidenced. The effects of transcription inhibition by actinomycin D and the results of nuclear run-on experiments suggest that H1 zero and H3.3 expression is regulated mainly at the posttranscriptional level. Concerning T3, only marginal effects were noticed, apart from up-regulation of both histone mRNAs at 2 days in culture. We propose one model for posttranscriptional regulation of the analyzed genes and discuss potential relationships to remodelling of chromatin.
Asunto(s)
Corteza Cerebral/metabolismo , Regulación de la Expresión Génica/fisiología , Histonas/genética , Neuronas/metabolismo , Procesamiento Postranscripcional del ARN , Animales , Northern Blotting , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , Medios de Cultivo , Dactinomicina/farmacología , Electroforesis en Gel de Poliacrilamida , Regulación de la Expresión Génica/efectos de los fármacos , Neuronas/efectos de los fármacos , Proteínas Nucleares/análisis , Ratas , Ratas Sprague-Dawley , SolubilidadRESUMEN
Synapsin I is a neuron-specific protein which is present in two isoforms, Ia and Ib. In the last few years this protein has been demonstrated to play a central role in the regulation of neurotransmitter release and synaptic plasticity. In this paper the developmental expression of this protein has been investigated in primary neuronal cultures from fetal rat brain cortices. The presence of thyroid hormone in the culture medium stimulates an early expression of the protein without exerting any effect at the level of mRNA transcription and accumulation. These observations implicate a T3-dependent regulation of this neuron-specific gene at the level of mRNA translation.
Asunto(s)
Corteza Cerebral/metabolismo , Expresión Génica , Neuronas/metabolismo , ARN Mensajero/biosíntesis , Sinapsinas/biosíntesis , Triyodotironina/farmacología , Animales , Northern Blotting , Western Blotting , Diferenciación Celular , Núcleo Celular/metabolismo , Células Cultivadas , Corteza Cerebral/citología , Feto , Expresión Génica/efectos de los fármacos , Cinética , Neuronas/citología , Neuronas/efectos de los fármacos , ARN Mensajero/análisis , Ratas , Factores de Tiempo , Transcripción Genética/efectos de los fármacosRESUMEN
Two overlapping rat cDNAs, covering a continuous region of 1107 base pairs, have been isolated and sequenced. The clones contain identical open reading frames, encoding a 136 amino acid long polypeptide which exhibits 100% identity to other mammalian H3.3 histone variants. We show that the inserts derive, in particular, from the H3.3B gene. We used these inserts and an insert from an H1(0) encoding clone, previously described (6), as probes to study the accumulation of mRNAs encoding the corresponding histone replacement variants (namely, H1(0) and H3.3) during rat brain development. We found that the concentration of both H1(0) and H3.3B mRNAs decreases from the embryonal day 18 (E18) to the postnatal day 10 (P10), with inverse correlation to protein accumulation.
Asunto(s)
Encéfalo/crecimiento & desarrollo , Histonas/genética , ARN Mensajero/metabolismo , Animales , Secuencia de Bases , Northern Blotting , Encéfalo/embriología , Encéfalo/metabolismo , ADN Complementario/química , ADN Complementario/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Variación Genética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Ratas , Mapeo Restrictivo , Alineación de SecuenciaRESUMEN
The aim of this review is to describe environmental requirements for survival of neuronal cells in culture, and secondly to survey the complex interplay between hormones, neurotrophic factors, transport- and extracellular matrix- proteins, which characterize the developmental program of differentiating neurons. An overall reconsideration of the literature in this vast field is above the limits of the present paper; since progress and refinement in the techniques of neuronal cell cultures have paralleled the advancement in Developmental Neurobiology, we will run instead through the main steps which form the conceptual framework of neuronal cell cultures.