RESUMEN
Due to its nootropic, neuroprotective, and immunomodulatory effects, the peptide Semax is utilized in the treatment of ischemic stroke. Our earlier RNA-Seq analysis of the transcriptome in an ischemic model of transient occlusion of the middle cerebral artery showed an increase in the mRNA levels of many proinflammatory genes, and the suppression of their induction by Semax. However, for many relevant genes, including Il1a, Il1b, Il6 and Tnfa, the levels of their expression were too low for detailed quantitative evaluation. Here we utilize qRT-PCR to analyze the effects of the Semax peptide on the expression of weakly expressed mRNAs encoding several proinflammatory mediators, and show that exposure to Semax leads to a statistically significant decrease in the Il1a, Il1b, Il6, Ccl3, and Cxcl2 mRNAs, which compensates for the increase in the transcription of these genes induced by ischemia-reperfusion. We conclude that the observed protective effect of Semax in the model of stroke may be due to its anti-inflammatory effects. We also discuss the limitations of the RNA-Seq when applied to quantifying less abundant transcripts as compared to the real-time RT-PCR method.
Asunto(s)
Isquemia Encefálica , Fármacos Neuroprotectores , Preparaciones Farmacéuticas , Hormona Adrenocorticotrópica/análogos & derivados , Animales , Encéfalo , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/genética , Isquemia , Fragmentos de Péptidos , ARN Mensajero/genética , Ratas , Ratas WistarRESUMEN
The importance of studying the action mechanisms of drugs based on natural regulatory peptides is commonly recognized. Particular attention is paid to the peptide drugs that contribute to the restoration of brain functions after acute cerebrovascular accidents (stroke), which for many years continues to be one of the main problems and threats to human health. However, molecular genetic changes in the brain in response to ischemia, as well as the mechanisms of protective effects of peptides, have not been sufficiently studied. This limits the use of neuroprotective peptides and makes it difficult to develop new, more efficient drugs with targeted action on brain functions. Transcriptome analysis is a promising approach for studying the mechanisms of the damaging effects of cerebral ischemia and neuroprotective action of peptide drugs. Beside investigating the role of mRNAs in protein synthesis, the development of new neuroprotection strategies requires studying the involvement of regulatory RNAs in ischemia. Of greatest interest are microRNAs (miRNAs) and circular RNAs (circRNAs), which are expressed predominantly in the brain. CircRNAs can interact with miRNAs and diminish their activity, thereby inhibiting miRNA-mediated repression of mRNAs. It has become apparent that analysis of the circRNA/miRNA/mRNA system is essential for deciphering the mechanisms of brain damage and repair. Here, we present the results of studies on the ischemia-induced changes in the activity of genes and peptide-mediated alterations in the transcriptome profiles in experimental ischemia and formulate the basic principles of peptide regulation in the ischemia-induced damage.
Asunto(s)
Encéfalo/metabolismo , Neuroprotección , Péptidos/metabolismo , Transcriptoma , Animales , Isquemia Encefálica/patología , Biología Computacional , Perfilación de la Expresión Génica , Humanos , MicroARNs/metabolismo , Neuronas/patología , ARN Circular/metabolismo , ARN Mensajero/metabolismoRESUMEN
Neurotrophins stimulate the regeneration of neural tissue after lesions. It is also known that the sources of neurogenesis and cerebral function recovery are predominantly located in subcortical brain structures. The effects of ischemia on the expression of genes that encode neurotrophins (Bdnf, Ngf, Nt-3) and their receptors (TrkB, TrkA, TrkC, p75) in brain structures outside the lesion site were studied 3, 24, and 72 h after irreversible unilateral occlusion of the middle cerebral artery in rats. Changes in the mRNA expression of these genes were assessed by relative quantification using real-time RT-PCR. Sham surgery was found to stimulate the expression of genes that encode neurotrophins (Bdnf, Ngf) and their receptor (p75). It has been shown that ischemia influenced the expression of neurotrophins (Bdnf, Ngf, Nt-3) and their receptors (TrkB, TrkA, TrkC, p75) in brain structures outside the lesion focus, including the contralateral hemisphere. The downregulation of Bdnf and TrkB transcripts and Ngf and TrkA upregulation in the contralateral cortex on the first day of ischemia obviously reflected stress response. On day 3, Nt-3 transcription increased in all investigated structures outside the lesion focus. In the contralateral hemisphere, relative levels of TrkA and TrkC mRNA expression increased, while p75 expression decreased. Presumably, the observed changes in gene transcription serve to facilitate neuroplasticity and neural tissue regeneration.
Asunto(s)
Isquemia Encefálica/metabolismo , Encéfalo/metabolismo , Regulación de la Expresión Génica , Polisacáridos/biosíntesis , Receptor de Factor de Crecimiento Nervioso/biosíntesis , Animales , Encéfalo/patología , Isquemia Encefálica/patología , Masculino , Ratas , Ratas WistarRESUMEN
The article is a review of the results of the study of the structural and functional organization of the human sphingomyelin synthase 1 gene (SGMS1) in Human Molecular Genetics Department of Institute of Molecular Genetics RAS. SGMS1 gene encodes an essential enzyme which is involved in the synthesis of sphingomyelin and diacylglycerol from phosphatidylcholine and ceramide, wich determines its participation in the regulation of intracellular vesicular transport, cholesterol metabolism, cell proliferation, apoptosis and other significant processes. Our research has shown that the SGMS1 gene is located on the chromosome 10, has a size of 320 kb and contains more than 20 exons. A detailed study of the SGMS1 gene's structure allowed us to identify the variety of its transcripts. mRNA isoforms with different fragments of 5R untranslated region (5R UTR) and encoding the full length protein, as well as transcripts resulting from alternative combinations of exons and containing the coding region of the gene and 3R UTR have been discovered. We have found new transcripts among the products of SGMS1 gene circular RNAs, which mostly contained sequences of multi-exon 5R UTR of the gene. They are conservative and predominantly expressed in the brain. Circular RNAs of SGMS1 gene had a large number of binding sites for a microRNA that may determine the functional significance of these molecules. The review describes the latest information about the structural and functional organization of the human gene SGMS1 as well as the features of its expression.
Asunto(s)
Regiones no Traducidas 5'/fisiología , Empalme Alternativo/fisiología , Cromosomas Humanos Par 10 , Regulación Enzimológica de la Expresión Génica/fisiología , Proteínas de la Membrana , MicroARNs , Proteínas del Tejido Nervioso , Transferasas (Grupos de Otros Fosfatos Sustitutos) , Cromosomas Humanos Par 10/genética , Cromosomas Humanos Par 10/metabolismo , Humanos , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , MicroARNs/genética , MicroARNs/metabolismo , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Transferasas (Grupos de Otros Fosfatos Sustitutos)/biosíntesis , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genéticaRESUMEN
The peptide preparation Semax has been effectively used for therapy of ischemic stroke. However, the mechanisms of its action are insufficiently understood and actively studied. The full-genome analysis of the transcriptome implemented in our recent work dem- onstrated that under conditions of focal ischemia of rat brain the Semax modified the profile of the transcription activity of many genes. In this case, the difference in the transcription levels of the gene encoding the protein transthyretin (Ttr) expression in rats under the pathological conditions of ischemia and in the presence of Semax was very high. High similarity between the effects of Ttr and coupled molecular systems with the Semax effects in ischemic stroke allowed us to suggest that the neuroprotection mechanisms of Semax (and, possibly, of other neuroprotection mechanisms of Semax) could be mediated by Ttr. In this review, we discussed the role of Ttr in CNS and its possible role in the neuroprotection mechanism of Semax.
Asunto(s)
Hormona Adrenocorticotrópica/análogos & derivados , Sistema Nervioso Central/metabolismo , Fármacos Neuroprotectores/farmacología , Fragmentos de Péptidos/farmacología , Prealbúmina/metabolismo , Hormona Adrenocorticotrópica/farmacología , Animales , Humanos , RatasRESUMEN
Sphingomyelin synthase 1 (SMS 1) catalyzes sphingomyelin biosynthesis in eukaryotic cells. We previously studied the structure of the human SGMS1 gene, which encodes the enzyme and its numerous transcripts. The tissue-specific expression of the transcripts was also described. Analysis of the SMS1 protein expression in human tissues using immunoblotting of tissue extracts prepared in the RIPA (Radio Immuno-Precipitation Assay) buffer revealed a weak signal in renal cortex, testis, lung, and no signal in placenta and lymphatic node. In this work, a new method of preparation of the tissue protein extracts enriched with SMS1 was suggested. The method based on the consecutive extraction with a buffer containing 0.05 and 1 mg/ml of the Quillaja saponaria saponin allowed SMS1 to be detected in all tissues tested. The SMS1 content in the saponin extract of kidney cortex is about 12-fold higher compared to the RIPA extraction procedure.
Asunto(s)
Bioquímica/métodos , Proteínas de la Membrana/análisis , Proteínas de la Membrana/química , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/química , Extractos de Tejidos/química , Transferasas (Grupos de Otros Fosfatos Sustitutos)/análisis , Transferasas (Grupos de Otros Fosfatos Sustitutos)/química , Tampones (Química) , Fraccionamiento Químico , Humanos , Immunoblotting/métodos , Corteza Renal/química , Quillaja/química , Saponinas/químicaRESUMEN
Sphingomyelin synthase 1 (SMS1) is an enzyme of vital importance which is responsible for the synthesis of sphingomyelin and diacylglycerol from phosphatidylcholine and ceramide in eukaryotic cells. Previously we have investigated in detail the structure of SGMS1 human gene and identified a lot of its transcripts. We revealed the isoforms of mRNA differing in the 5'-UTR and coding the full-length protein, and also the transcripts arising from alternative combination of the exons localized in the coding region of the gene and 3'-UTR. From the results of computer analysis it follows that the synthesis of transcripts differing in the 5'-UTR is enabled by the different promoters of SGMS1 gene. It has been found in the present work by the method of real-time PCR that the content of five alternative transcripts of this gene, differing in the 5'-UTR, is substantially dissimilar among human tissues. In all the investigated tissues those transcripts are presented most prominently whose synthesis takes place under the control of the distal promoter including exon 1. In lesser extent are presented the transcripts including 5'-end exons whose synthesis is enabled by the promoters localized in introns of this gene. The differential level of content of SGMS1 gene transcripts, differing in the 5'-UTR, indicates that the use of the alternative promoters is tissue-specific and apparently strictly regulated. The structural organization of 5'-UTR variants of SGMS1 transcripts, directed by alternative promoters, is substantially different; this can provide regulation of the gene functioning on post-transcriptional level.
Asunto(s)
Regulación Enzimológica de la Expresión Génica/fisiología , Intrones/fisiología , Proteínas de la Membrana , Proteínas del Tejido Nervioso , Regiones Promotoras Genéticas/fisiología , Transcripción Genética/fisiología , Transferasas (Grupos de Otros Fosfatos Sustitutos) , Regiones no Traducidas 3'/fisiología , Regiones no Traducidas 5'/fisiología , Femenino , Humanos , Masculino , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Especificidad de Órganos/fisiología , Transferasas (Grupos de Otros Fosfatos Sustitutos)/biosíntesis , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genéticaRESUMEN
The investigation of molecular mechanisms contributing to cancer progression is the burning problem ofcurrent research. Considerable attention has been given to the study of gene expression in cancer cells. Sphingomyelin synthase 1 gene (SGMS1) is one of the genes whose expression can be altered in cancer. SMS1 enzyme, encoded by this gene, catalyzes the synthesis of sphingomyelin and diacylglycerol from phosphatidylcholine and ceramide. SMS1 may maintain the balance between cell death and survival by regulating the formation of the pro-apoptotic mediator ceramide and anti-apoptotic mediator diacylglycerol. In addition, the changes in sphingomyelin level and sphingomyelin synthase activity have been observed in cancers of many tissues. However the peculiarities of SGMS1 gene transcription have been insufficiently explored. In this work the expression of transcripts of SGMS1 has been investigated by the method of Real Time PCR in matched pairs of samples of human lung and oesophagus cancer and adjacent tissues without pathology. A significant decrease in SMS1 transcripts expression has been found in samples of human lung cancer. At the same time, in the samples of human oesophagus cancer and adjacent tissue, expression of SMS1 transcripts varies insignificantly: it is increased in 7 and decreased in 5 of 15 samples. The obtained results indicate that SGMS1 gene is differently expressed in cancers of different genesis.
Asunto(s)
Neoplasias Esofágicas/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Transcripción Genética , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genéticaRESUMEN
Biologically active regulatory peptide, tripeptide Pro-Gly-Pro (PGP) was used as C-terminal fragment for peptide drugs Semax and Selank. In recent years the independent effects of PGP were observed. The question was raised, whether PGP contributes to the effects ofpeptide drugs containing PGP as a fragment. The genome-wide analysis was performed to investigate the influence of PGP on the transcriptome of ischemic rat brain cortex tissues. The gene expression alterations caused by the action of the tripeptide PGP were compared with the gene expression of the control group "ischemia" at 3 and 24 h after permanent occlusion of left middle cerebral artery. The altered expression was detected for 29 genes at 3 h and 57--at 24 h. The proteins encoded by these genes have variety of functions: cytokines, transport proteins, transcription factors, transmembrane receptors, etc. Biological processes, which are related to the genes with altered expression, were distinguished. The influence of PGP on the diversity of biological processes in different systems of the organism is demonstrated for the first time. The process "Immune response" was the most statistically notable at 24 h after occlusion. The expression of the immune system genes was predominately down regulated.
Asunto(s)
Isquemia Encefálica/genética , Trastornos Cerebrovasculares/genética , Regulación de la Expresión Génica/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Oligopéptidos/farmacología , Prolina/análogos & derivados , Transcriptoma , Animales , Isquemia Encefálica/inmunología , Isquemia Encefálica/patología , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Trastornos Cerebrovasculares/inmunología , Trastornos Cerebrovasculares/patología , Citocinas/genética , Citocinas/inmunología , Perfilación de la Expresión Génica , Inmunidad Innata/efectos de los fármacos , Masculino , Prolina/farmacología , Ratas , Ratas Wistar , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/inmunología , Factores de Transcripción/genética , Factores de Transcripción/inmunologíaRESUMEN
Vascular endothelial growth factor (VEGFA) is a hypoxia-inducible signal glycoprotein. VEGFA causes vascular endothelial cell growth and proliferation, that leads to the regeneration of vascular network in brain regions damaged by ischemia. However, this protein is involved in processes of inflammation and edema in early stages of ischemia. Synthetic peptide semax shows neuroprotective and anti-inflammatory properties and is actively used in the treatment of ischemia.We have previously shown that semax reduces vascular injury and activates the mRNA synthesis of neurotrophins and their receptors under global cerebral ischemia in rats. Here we have analyzed the effects of semax and its C-terminal Pro-Gly-Pro tripeptide upon Vegfa mRNA expression in different rat brain regions after common carotid artery occlusion. The animals were decapitated 30 min, 1, 2, 4, 8, 12, 24 h after the operation. It was shown that ischemia increases levels of Vegfa mRNA in the rat brain of animals (4 h after the occlusion--in the cerebellum, cerebral cortex and hippocampus, 8 h--in the cortex and hippocampus, and 24 h in the cortex). Semax treatment reduces Vegfa mRNA levels in the frontal cortex (4, 8 and 12 h after the occlusion) and hippocampus of ischemic rats (2 and 4 h). Effect of PGP on the Vegfa gene expression was almost negligible. Our results showed that semax prevents activating effect ofhypoxia on the Vegfa gene expression in early stages of global ischemia. Furthermore, increase in the level of mRNA Vegfa in the hippocampus (24 h after occlusion) perhaps reflects neuroprotective properties of this drug.
Asunto(s)
Hormona Adrenocorticotrópica/análogos & derivados , Isquemia Encefálica/metabolismo , Encéfalo/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas del Tejido Nervioso/biosíntesis , Fármacos Neuroprotectores/farmacología , Oligopéptidos/farmacología , Fragmentos de Péptidos/farmacología , Prolina/análogos & derivados , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Hormona Adrenocorticotrópica/farmacología , Animales , Encéfalo/patología , Isquemia Encefálica/patología , Masculino , Prolina/farmacología , Ratas , Ratas WistarRESUMEN
Neurotrophins regulate key function of nervous tissue cells. Analysis of neurotrophins mRNA expression is an appropriate tool to assess therapeutic efficiency of the anti-stroke drugs. We have analyzed the effect of synthetic peptide semax and its C-terminal Pro-Gly-Pro tripeptide upon mRNAs expression of neurotrophins Ngf, Bdrf, Nt-3 and their receptors TrkA, TrkB, TrkC, p75 in rat frontal lobes, hippocampus and cerebellum after bilateral common carotid artery occlusion. The animals were decapitated 30 min, 1, 2, 4, 8, 12, 24 h after the operation. The mRNA expression of neurotrophins and their receptors was assessed by relative quantification using real-time RT-PCR. Our showed that ischemia causes a significant decrease in gene expression in the hippocampus. Semax and PGP affected the expression of neurotrophins and their receptors predominantly in the frontal cortex and hippocampus of the ischemized animals. In the frontal cortex, Semax treatment resulted in a decrease of mRNA level of receptors, while PGP treatment increased the level of these mRNA. Maximal neuroprotective effect of both peptides has been observed in the hippocampus 12 h after occlusion. A decrease of gene expression of neurotrophins and their receptors caused by the occlusion was overcome by Semax and PGP. These results clarify the semax mechanism of and present certain features of mRNA's expression of neurotrophins and their receptors in experimental conditions.
Asunto(s)
Hormona Adrenocorticotrópica/análogos & derivados , Isquemia Encefálica/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Hipocampo/efectos de los fármacos , Factores de Crecimiento Nervioso/genética , Oligopéptidos/farmacología , Fragmentos de Péptidos/farmacología , Prolina/análogos & derivados , Hormona Adrenocorticotrópica/farmacología , Animales , Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Hipocampo/metabolismo , Masculino , Factor de Crecimiento Nervioso/genética , Factor de Crecimiento Nervioso/metabolismo , Prolina/farmacología , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptor de Factor de Crecimiento Nervioso/genética , Receptor de Factor de Crecimiento Nervioso/metabolismo , Receptor trkA/genética , Receptor trkA/metabolismo , Receptor trkB/genética , Receptor trkB/metabolismo , Receptor trkC/genética , Receptor trkC/metabolismoAsunto(s)
Hormona Adrenocorticotrópica/análogos & derivados , Isquemia Encefálica/metabolismo , Corteza Cerebral/metabolismo , Factores de Crecimiento Nervioso/metabolismo , Oligopéptidos/administración & dosificación , Fragmentos de Péptidos/administración & dosificación , Prolina/análogos & derivados , Receptores de Factor de Crecimiento Nervioso/metabolismo , Hormona Adrenocorticotrópica/administración & dosificación , Animales , Corteza Cerebral/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Expresión Génica/efectos de los fármacos , Masculino , Fármacos Neuroprotectores/administración & dosificación , Prolina/administración & dosificación , Ratas , Ratas WistarRESUMEN
Neurotrophins are structurally related growth polypeptide factors that play an essential role in the development and functioning of the vertebrate nervous system. They provide forming and survival of different neuron populations of the central and peripheral nervous system. Neurotrophins are also involved in the processes of higher nervous activity. Neurotrophins are active not only in the nervous system; these universal trophic factors are important for the development, proliferation, and maintaining of different tissues including tumor tissues. Changes in the neurotrophin signaling system are significant for the pathogenesis of malignancies at the initiation stage as well as during the tumor progression. Neurotrophins and their receptors are complex multi-component system controlled in a very complicated manner. This system can affect the cells and tissues in different ways; the final results of neurotrophin action vary from cell maintenance and survival to apoptosis. Differences in mechanisms and results of the neurotrophin action depend on the cell and tissue type in which the system works. The effects of the neurotrophin signaling are especially variable in different malignancies. In the review we summarize the information on the neurotrophin signaling in various tumors and demonstrate its contribution to the disease course.
Asunto(s)
Neoplasias/metabolismo , Factores de Crecimiento Nervioso/fisiología , Receptores de Factor de Crecimiento Nervioso/fisiología , Animales , Antineoplásicos/farmacología , Transformación Celular Neoplásica/metabolismo , Progresión de la Enfermedad , Humanos , Neoplasias/diagnóstico , Neoplasias/terapia , Factor de Crecimiento Nervioso/antagonistas & inhibidores , Factor de Crecimiento Nervioso/farmacología , Factores de Crecimiento Nervioso/antagonistas & inhibidores , Receptor trkA/metabolismo , Receptores de Factor de Crecimiento Nervioso/antagonistas & inhibidores , Transducción de SeñalRESUMEN
Brain-specific human genes were studied over the recent years in the Department of Molecular Bases of Human Genetics, Institute of Molecular Genetics. Clones Hfb1, Hmob3, and Hmob33 were selected from human brain cDNA libraries by differential screening. The clones were sequenced, mapped, and tested for expression in various human tissues. In vitro and in silico experiments identified Hfb1 as an earlier unknown complexin 2 gene (CPLX2) fragment, which codes for the large 3'-untranslated region of the CPLX2 mRNA. Hmob3 proved to correspond to an earlier unknown fragment of the large 3'-untranslated region of the human MAP1B mRNA. With Hfb1 and Hmob3, new terminal exons were revealed and exact structures established for CPLX2 and MAP1B. Hmob33 was identified as a fragment of the 3'-terminal exon of a new gene, MOB, which codes for a thus far unknown evolutionarily conserved transmembrane protein. The structure of the deduced protein product was analyzed.
Asunto(s)
Encéfalo/fisiología , Proteínas de Unión al ADN/genética , Proteínas de la Membrana , Proteínas del Tejido Nervioso/genética , Factores de Transcripción/genética , Regiones no Traducidas 3' , Proteínas Adaptadoras del Transporte Vesicular , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Clonación Molecular , Secuencia Conservada , Proteínas de Unión al ADN/química , Evolución Molecular , Exones , Secuencias Hélice-Asa-Hélice , Humanos , Proteínas Asociadas a Microtúbulos/genética , Datos de Secuencia Molecular , Especificidad de Órganos , Filogenia , ARN Mensajero , Análisis de Secuencia de ADN , Factores de Transcripción/química , Transferasas (Grupos de Otros Fosfatos Sustitutos)RESUMEN
A novel evolutionary conserved human MOB gene of seven exons is predicted on the chromosome 10. MOB is supposed to express predominately in brain. At least three types of MOB transcripts are proposed. A protein encoded by MOB is five-pass transmembrane molecule.
Asunto(s)
Cromosomas Humanos Par 10/genética , Bulbo Raquídeo/fisiología , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso , Secuencia de Aminoácidos , Animales , Northern Blotting , Mapeo Cromosómico , Clonación Molecular , Secuencia Conservada/genética , Mapeo Contig , ADN Complementario , Exones/genética , Humanos , Técnicas In Vitro , Proteínas de la Membrana/química , Ratones , Datos de Secuencia Molecular , Reconocimiento de Normas Patrones Automatizadas , Filogenia , Análisis de Secuencia de ADN , Transferasas (Grupos de Otros Fosfatos Sustitutos)RESUMEN
A genomic clone hybridizing with brain-specific sequence Hfb1 was isolated from a chromosome 5 consmid library. Hfb1 proved to correspond to a new gene exon which codes for a large 3'-untranslated region of the mRNA for synaptic protein complexin 2. Together with the 985-nt Hfb1 cDNA (EMBL Y15167) isolated previously from a cDNA library of the frontal cerebral cortex, the primary structure was established for genomic clone Ghfb sized more than 4 kb. A GenBank search revealed complete identity of the 5' end of Ghfb and the 3'-untranslated region (878-933) of the human complexin 2 mRNA. Large transcripts with the 5' end corresponding to the complexin 2 mRNA and the 3' end to Ghfb were detected in total mRNA of the human brain by means of RT-PCR. The size of the 3'-untranslated region of the human complexin 2 mRNA was estimated at 4 kb.
Asunto(s)
Regiones no Traducidas 3' , Encéfalo/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas Adaptadoras del Transporte Vesicular , Secuencia de Bases , Mapeo Cromosómico , Cromosomas Humanos Par 5 , ADN Complementario , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The cDNA libraries in gt10 were constructed from total poly(A)+RNA of human forebrain cortex, cerebellar cortex and medulla oblongata. We selected the clones which gave hybridization signal with brain cDNA only, or gave no signal from these libraries. Expression pattern and structure of two brain-specific clones Hfb1 from forebrain library and Hmob3 from medulla oblongata library were analyzed in detail. Hfb1 hybridized to two different transcripts (about 5 and 2 kb) from frontal cortex, but to a single (longest) from cerebellum. Hfb1 sequence includes 958 nucleotides. Comparison of Hfb1 with the Gene Bank revealed no homology with the sequences present in the Bank. At 3'-end there is poly(A) tail of 24 bases, there is the AATCAA sequence 55 nucleotides upstream which probably serves as a polyadenylation signal. However, AATCAA directs polyadenylation in vitro with very low efficiency. We found no open reading frame in the clone and this is in agreement with the data indicating that brain-specific RNAs has extremely long 3'-untranslated regions. Hmob3 was partially sequences. We compared its primary structure with the sequences from the Gene Bank and revealed no homology. Hmob3 expresses in different parts of human brain and in sceletal muscle but does not express in other tissues.
Asunto(s)
Encéfalo/metabolismo , ADN , Secuencia de Bases , Northern Blotting , Southern Blotting , Expresión Génica , Biblioteca Genómica , Humanos , Datos de Secuencia Molecular , Poli A/metabolismo , ARN/metabolismo , ARN MensajeroRESUMEN
Evolutionary relationships of the IncN plasmid R15 and other broad host range plasmids (IncN plasmids N3 and R46, IncP plasmids RP4 and R906, IncW plasmids Sa and R388) were studied by Southern blot hybridization technique. The IncN plasmids were shown to harbour homologous determinants for replication and conjugation. No homology was found between the rep and tra genes in R15 and in the IncW and IncP plasmids, respectively. The second rep region of the N3 plasmid is distinctive from the corresponding determinants in the IncN plasmids. Homology was demonstrated for the plasmid genes that mediate restriction and modification in R15 and N3, mercury resistance in R15 and R906, sulfanilamide resistance in R15, N3, R46, Sa, R388, and R906, streptomycin resistance in R15, R46 and Sa. The latter genes are different from the R906 SmR gene. In addition to the three known mobile elements in the plasmid R15, the fourth one (IS46) that is a part of the transposon Tn2353 was identified in this study. Besides, the third copy of this insertion sequence was found in the N3 plasmid.