RESUMO
The golli proteins, products of the myelin basic protein gene, are widely expressed in oligodendrocyte progenitor cells and neurons during the postnatal development of the brain. While golli appears to be important for oligodendrocyte migration and differentiation, its function in neuronal development is completely unknown. We have found that golli proteins function as new and novel modulators of voltage-operated Ca(++) channels (VOCCs) in neurons. In vitro, golli knock-out (KO) neurons exhibit decreased Ca(++) influx after plasma membrane depolarization and a substantial maturational delay. Increased expression of golli proteins enhances L-type Ca(++) entry and processes outgrowth in cortical neurons, and pharmacological activation of L-type Ca(++) channels stimulates maturation and prevents cell death in golli-KO neurons. In situ, Ca(++) influx mediated by L-type VOCCs was significantly decreased in cortical and hippocampal neurons of the golli-KO brain. These Ca(++) alterations affect cortical and hippocampal development and the proliferation and survival of neural progenitor cells during the postnatal development of the golli-KO brain. The CA1/3 sections and the dentate gyrus of the hippocampus were reduced in the golli-KO mice as well as the density of dendrites in the somatosensory cortex. Furthermore, the golli-KO mice display abnormal behavior including deficits in episodic memory and reduced anxiety. Because of the expression of the golli proteins within neurons in learning and memory centers of the brain, this work has profound implication in neurodegenerative diseases and neurological disorders.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Hipocampo/citologia , Proteína Básica da Mielina/metabolismo , Neurônios/metabolismo , Animais , Ansiedade/metabolismo , Ansiedade/fisiopatologia , Comportamento Animal , Sinalização do Cálcio , Diferenciação Celular , Proliferação de Células , Separação Celular , Sobrevivência Celular , Camundongos Knockout , Atividade Motora , Neurogênese , Neurônios/citologiaRESUMO
Transgenic mice were generated to permit the targeted ablation of cortical preplate cells at the time they are born. In these mice, the 1.3 kb golli promoter of the myelin basic protein gene was used to drive the herpes simplex virus thymidine kinase (TK) transgene in cortical preplate cells. Heterozygous transgenic pairs were bred, and pregnant dams were treated with ganciclovir at embryonic days 11-12 to ablate preplate cells at the time the preplate was forming. This paradigm exposed control (TK-) and experimental (TK+) littermates to exactly the same conditions. Embryological ablation of preplate cells led to an early disruption of the radial glial framework and subplate structure in the developing cortex and dramatically altered the cellular lamination and connectivity of the cortical plate. The disturbed radial glial network contributed to an impaired radial migration of neurons into the cortical plate from the ventricular zone. The cortical plate became dyslaminated, and there was a substantial reduction in short- and long-range cortical projections within the cortex and to subcortical regions. Cell death within the cortical plate and the proliferative zones was substantially increased in the ablated animals. After birth, a cortical lesion developed, which became exacerbated with the secondary onset of hydrocephaly in the second postnatal week. The results underscore the critical importance of the preplate in cortex formation, mediated through its guidance of the formation of radial glial scaffolding, subsequent neuronal migration into the incipient cortical plate, and the final arrangement of its vertical organization and cellular connectivity.
Assuntos
Córtex Cerebral/embriologia , Estruturas Embrionárias/embriologia , Neurônios/efeitos dos fármacos , Animais , Bromodesoxiuridina , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Movimento Celular/efeitos dos fármacos , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Estruturas Embrionárias/citologia , Estruturas Embrionárias/efeitos dos fármacos , Ganciclovir/farmacologia , Hidrocefalia/induzido quimicamente , Hidrocefalia/genética , Hidrocefalia/patologia , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Transgênicos , Modelos Animais , Proteína Básica da Mielina/genética , Malformações do Sistema Nervoso/induzido quimicamente , Malformações do Sistema Nervoso/genética , Malformações do Sistema Nervoso/patologia , Neuroglia/citologia , Neuroglia/efeitos dos fármacos , Neurônios/citologia , Regiões Promotoras Genéticas/genética , Simplexvirus/genética , Timidina Quinase/biossíntese , Timidina Quinase/genéticaRESUMO
Recently, we described the identification of novel streptococcal superantigens (SAgs) by mining the Streptococcus pyogenes M1 genome database at Oklahoma University. Here, we report the cloning, expression, and functional analysis of streptococcal pyrogenic exotoxin (SPE)-J and another novel SAg (SPE-I). SPE-I is most closely related to SPE-H and staphylococcal enterotoxin I, whereas SPE-J is most closely related to SPE-C. Recombinant forms of SPE-I and SPE-J were mitogenic for PBL, both reaching half maximum responses at 0.1 pg/ml. Evidence from binding studies and cell aggregation assays using a human B-lymphoblastoid cell line (LG-2) suggests that both toxins exclusively bind to the polymorphic MHC class II beta-chain in a zinc-dependent mode but not to the generic MHC class II alpha-chain. The results from analysis by light scattering indicate that SPE-J exists as a dimer in solution above concentrations of 4.0 mg/ml. Moreover, SPE-J induced a rapid homotypic aggregation of LG-2 cells, suggesting that this toxin might cross-link MHC class II molecules on the cell surface by building tetramers of the type HLA-DRbeta-SPE-J-SPE-J-HLA-DRbeta. SPE-I preferably stimulates T cells bearing the Vbeta18.1 TCR, which is not targeted by any other known SAG: SPE-J almost exclusively stimulates Vbeta2.1 T cells, a Vbeta that is targeted by several other streptococcal SAgs, suggesting a specific role for this T cell subpopulation in immune defense. Despite a primary sequence diversity of 51%, SPE-J is functionally indistinguishable from SPE-C and might play a role in streptococcal disease, which has previously been addressed to SPE-C.
Assuntos
Proteínas de Bactérias , Exotoxinas/imunologia , Proteínas de Membrana , Streptococcus pyogenes/imunologia , Superantígenos/imunologia , Sequência de Aminoácidos , Dimerização , Exotoxinas/química , Exotoxinas/genética , Exotoxinas/metabolismo , Regulação Bacteriana da Expressão Gênica/imunologia , Genes Bacterianos , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Ligantes , Ativação Linfocitária/imunologia , Dados de Sequência Molecular , Ligação Proteica/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/biossíntese , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Streptococcus pyogenes/química , Streptococcus pyogenes/genética , Superantígenos/química , Superantígenos/genética , Superantígenos/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Zinco/química , Zinco/metabolismoRESUMO
The mouse oligodendrocyte cell lines, N19 and N20.1, were used as sources of potential stage-specific RNA in order to construct a subtraction library enriched in cDNAs expressed early in the oligodendrocyte (OL) lineage. From this library, 23 clones were examined and three were examined in most detail. The mRNAs of the three library clones were preferentially expressed in the N19 (progenitor) compared to the N20.1 (immature) OL line. One of these corresponded to the intermediate filament protein cytokeratin K19, which has not been reported to be expressed in OLs previously. Another was identified as the mouse homolog of T-cadherin, previously reported not to be present in OLs. Antisera raised against a T-cadherin peptide indicated the protein colocalized with the OL lineage markers A(2)B(5), A007, and 01 in mouse primary glial cultures. However, small round cells resembling OL precursors labeled intensely with T-cadherin, but were negative for the other markers, suggesting that this gene might be expressed earlier in the lineage. In early postnatal brain, in addition to the expected neuronal tracts, the T-cadherin antibody labeled small bipolar cells, approximately 8-10 microm in diameter, in white matter tracts. These cells had the morphology of OLs or their precursors and were identified within the cerebellar white matter and the corpus callosum, regions rich in OLs. The third clone, 3g5, was homologous to the P8 clone isolated from rat pancreas. It encoded an 80-amino-acid polypeptide with a protein kinase C domain suggesting a possible role in signal transduction. Antisera to this peptide also colocalized 3g5 with cells expressing A(2)B(5), A007, and 01 in culture and in cells within white matter tracts which had the same morphology as those labeled by T-cadherin in these regions. In addition to these, beta(10) thymosin and mevalonate kinase clones were also isolated from the screen.
Assuntos
Diferenciação Celular/genética , Linhagem da Célula/genética , Sistema Nervoso Central/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento/genética , Genes/fisiologia , Oligodendroglia/metabolismo , Células-Tronco/metabolismo , Animais , Linhagem Celular Transformada , Células Cultivadas , Sistema Nervoso Central/citologia , Sistema Nervoso Central/metabolismo , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Biblioteca Gênica , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Oligodendroglia/citologia , Homologia de Sequência de Aminoácidos , Células-Tronco/citologiaRESUMO
The golli products of the myelin basic protein gene have been shown to be expressed in mouse thymus and brain. The full repertoire of thymic cell types expressing golli products has not yet been determined, although immunoreactivity has been found in some macrophages. We have analyzed the cellular expression of golli mRNAs and proteins in the thymus. The results showed that MTS5(+) cortical/MTS10(+) medullary epithelial cells and NLDC145(+) dendritic cells did not express golli, while some macrophages did exhibit strong immunoreactivity. GOLLI: mRNAs were not detected in macrophages by in situ hybridization. Thymocytes expressed significant levels of golli mRNAs and proteins by in situ hybridization and immunohistochemistry. Interestingly, golli immunoreactivity varied with thymocyte stage of differentiation. For example, CD4(-)CD8(-) (double-negative) thymocytes expressed relatively high levels of golli. Upon further differentiation into CD4(-)CD8(-) (double-positive) thymocytes, golli protein expression declined dramatically. When thymocytes developed into CD8(-) or CD4(+) (single-positive) thymocytes, golli protein expression increased again, but it never achieved the levels found in double-negative thymocytes. Thus, the altered levels of expression of golli proteins in developing thymocytes correlated with the transitions from double-negative to double-positive and double-positive to single-positive stages. The lack of significant golli expression in thymic stromal cells may offer an alternative explanation for the mechanism of inefficient negative selection of those autoreactive thymocytes with specificity for myelin basic proteins.
Assuntos
Regulação da Expressão Gênica/imunologia , Proteína Básica da Mielina/biossíntese , Proteína Básica da Mielina/genética , Linfócitos T/metabolismo , Timo/metabolismo , Animais , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Núcleo Celular/química , Núcleo Celular/imunologia , Núcleo Celular/metabolismo , Citoplasma/química , Citoplasma/imunologia , Citoplasma/metabolismo , Tolerância Imunológica , Imuno-Histoquímica , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/biossíntese , Células Estromais/química , Células Estromais/imunologia , Células Estromais/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T/química , Linfócitos T/imunologia , Timo/citologia , Timo/imunologiaRESUMO
The myelin proteolipid protein (PLP) gene (i.e., the PLP/DM20 gene) has been of some interest because of its role in certain human demyelinating diseases, such as Pelizaeus-Merzbacher disease. A substantial amount of evidence, including neuronal pathology in knock-out and transgenic animals, suggests the gene also has functions unrelated to myelin structure, but the products of the gene responsible for these putative functions have not yet been identified. Here we report the identification of a new exon of the PLP/DM20 gene and at least two new products of the gene that contain this exon. The new exon, located between exons 1 and 2, is spliced into PLP and DM20 mRNAs creating a new translation initiation site that generates PLP and DM20 proteins with a 12 amino acid leader sequence. This leader sequence appears to target these proteins to a different cellular compartment within the cell bodies of oligodendrocytes and away from the myelin membranes. Furthermore, these new products are also expressed in a number of neuronal populations within the postnatal mouse brain, including the cerebellum, hippocampus, and olfactory system. We term these products somal-restricted PLP and DM20 proteins to distinguish them from the classic PLP and DM20 proteolipids. They represent putative candidates for some of the nonmyelin-related functions of the PLP/DM20 gene.
Assuntos
Encéfalo/metabolismo , Éxons , Regulação da Expressão Gênica no Desenvolvimento , Proteína Proteolipídica de Mielina/genética , Proteínas do Tecido Nervoso , Neurônios/metabolismo , Oligodendroglia/metabolismo , Envelhecimento , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/embriologia , Encéfalo/crescimento & desenvolvimento , Células Cultivadas , Esclerose Cerebral Difusa de Schilder/genética , Biblioteca Genômica , Humanos , Íntrons , Camundongos , Dados de Sequência Molecular , Biossíntese de Proteínas , Isoformas de Proteínas/genética , Sinais Direcionadores de Proteínas/genética , Transcrição GênicaRESUMO
Tobacco mosaic virus (TMV) induces the hypersensitive response (HR) in tobacco plants containing the N gene. This defence response is characterized by cell death at the site of virus infection and inhibition of viral replication and movement. A previous study indicated that a portion of the TMV replicase containing a putative helicase domain is involved in HR induction. Here, this observation is confirmed and extended by showing that non-viral expression of a 50 kDa TMV helicase fragment (p50) is sufficient to induce the N-mediated HR in tobacco. Like the HR elicited by TMV infection, transgenic expression of p50 induces a temperature-sensitive defence response. We demonstrate that recombinant p50 protein has ATPase activity, as suggested by the presence of conserved sequence motifs found in ATPase/helicase enzymes. A point mutation that alters one of these motifs abolishes ATPase activity in vitro but does not affect HR induction. These results suggest that features of the TMV helicase domain, independent of its enzymatic activity, are recognized by N-containing tobacco to induce TMV resistance.
Assuntos
Nicotiana/virologia , Plantas Tóxicas , RNA Helicases/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Vírus do Mosaico do Tabaco/enzimologia , Adenosina Trifosfatases/metabolismo , Genes de Plantas , Genes Virais , Plantas Geneticamente Modificadas , RNA Helicases/química , RNA Helicases/genética , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , Rhizobium/genética , Temperatura , Nicotiana/enzimologia , Nicotiana/genética , Vírus do Mosaico do Tabaco/patogenicidadeRESUMO
The interaction between tobacco mosaic virus (TMV) and tobacco harbouring the N gene is a classical system for studying gene-for-gene interactions in disease resistance. The N gene confers resistance to TMV by mediating defence responses that function to limit viral replication and movement. We isolated the N gene and determined that N belongs to the nucleotide-binding-site-leucine-rich-repeat (NBS-LRR) class of plant disease resistance genes, and encodes both full-length and truncated proteins. Sequence homologies and mutagenesis studies indicated a signalling role for the N protein similar to that seen for proteins involved in defence responses in insects and mammals. The N gene confers resistance to TMV in transgenic tomato, demonstrating the use of the NBS-LRR class of disease resistance genes in engineering crop resistance. From the pathogen side of this interaction, the TMV 126 kDa replicase protein has been implicated as the avirulence factor that triggers N-mediated defence responses. We employed Agrobacterium-mediated expression strategies to demonstrate that expression of the putative helicase region of the replicase protein is sufficient to elicit N-mediated defences. The thermosensitivity of the N-mediated response to TMV is retained when induced by expression of this replicase fragment. Thus, both components of this gene-for-gene interaction are now available for studies that address the molecular mechanisms involved in N-mediated TMV resistance.
Assuntos
Nicotiana/genética , Proteínas de Plantas/genética , Plantas Tóxicas , Vírus do Mosaico do Tabaco/genética , Técnicas de Transferência de Genes , Imunidade Inata/genética , Solanum lycopersicum , Nicotiana/virologiaRESUMO
Resistance in tomato line Hawaii 7998 as well as in several nonhost plants to Xanthomonas campestris pv. vesicatoria tomato strain (XcvT) is mediated in part by the avirulence gene avrRxv. Analysis of growth of wild-type and avrRxv deletion strains indicates that avrRxv plays a crucial role in the ability of XcvT 92-14 to induce resistance on Hawaii 7998. We used avrRxv reporter gene fusions and Northern (RNA) blot analysis to test several growth environments for inductive potential. We found that avrRxv is constitutively expressed at high levels and that growth in planta, in tobacco conditioned medium, and in hrp-inductive medium XVM2 did not affect the high levels of expression. In addition, hrp structural and regulatory mutant backgrounds had no effect. We mutated the bipartite plant inducible promoter (PIP)-box sequence and found that avrRxv activity appears to be independent of an intact PIP-box element. We present the sequence of the avrRxv homologue called avrBsT and align the six AvrRxv host interaction factor family members including mammalian pathogen virulence factors YopJ and YopP from Yersinia spp. and AvrA from Salmonella typhimurium, and open reading frame Y4LO with unknown function from the symbiont Rhizobium sp.
Assuntos
Proteínas de Bactérias/genética , Genes Bacterianos , Solanum lycopersicum/microbiologia , Xanthomonas campestris/genética , Xanthomonas campestris/patogenicidade , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA/genética , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Genes Reporter , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fenótipo , Regiões Promotoras Genéticas , Homologia de Sequência de Aminoácidos , Virulência/genética , Xanthomonas campestris/crescimento & desenvolvimentoRESUMO
The cellular localization of the dopaminergic D2 receptor (D2R) mRNA and protein was determined during postnatal development, from birth to 35 days, in the rat neostriatum by in situ hybridization histochemistry and immunohistochemistry. To localize and identify more precisely the morphology of cells expressing the D2R mRNA, nonradioactive, digoxigenin in situ hybridization was performed. Throughout this period of development, D2R mRNA and protein were widely expressed by neostriatal cells, adjoining forebrain cells and small cellular processes. Within morphologically identifiable neurons, the expression of the D2 receptor appeared to occur after cell division ceased. D2R gene expression appeared during neuronal migration and followed the developmental pattern of neuronal settling within the neostriatum. Both D2R mRNA and protein appeared to colocalize in neostriatal cells and the labeling of both appeared to accumulate within the cells progressively with age. The structural phenotypes of neostriatal neurons bearing D2R mRNA and protein were diverse throughout postnatal development. The most frequently stained cells were a heterogeneous group of medium spiny and aspiny neurons. Large cells corresponding to aspiny neurons were less frequently stained. Both phenotypes exhibited considerable postnatal growth of their cell bodies. In addition to neurons, other cell types were also observed to express the D2R mRNA and protein over the developmental period studied. These other cells included patches of ciliated ependymal cells lining the lateral ventricles and many interfascicular oligodendroglia of forebrain fiber tracts. These results demonstrate the unexpected expression of the dopaminergic D2 receptor in non-neuronal cells within the brain. They provide a novel morphologic suggestion that the dopaminergic D2 receptor may support unrecognized, nonsynaptic functions in specific non-neuronal cell populations in the nervous system.
Assuntos
Neostriado/química , Prosencéfalo/química , Receptores de Dopamina D2/genética , Animais , Epêndima/citologia , Epêndima/metabolismo , Feminino , Histocitoquímica , Imuno-Histoquímica , Hibridização In Situ , Masculino , Morfogênese , Neostriado/embriologia , Neostriado/crescimento & desenvolvimento , Neurônios/metabolismo , Oligodendroglia/metabolismo , Prosencéfalo/embriologia , Prosencéfalo/crescimento & desenvolvimento , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-DawleyRESUMO
Demyelinating diseases, such as multiple sclerosis (MS) in man or experimental allergic encephalomyelitis (EAE) in rodents, may include an associated immune response directed against myelin protein antigens such as the proteolipid protein (PLP) and the myelin basic protein (MBP). Development of an immune response has been attributed, in part, to the sequestration of central nervous system antigens behind the blood-brain barrier. Recently, we identified a novel gene, the golli gene, which overlaps the mbp gene. The Golli transcription unit produces a family of mRNAs, and their corresponding proteins possess MBP epitopes known to be encephalitogenic in EAE. Transcription of the golli gene was detected in immune system tissue. Therefore, we wished to determine whether genes that encode the two major myelin protein components, PLP and MBP, were expressed in the human thymus. Our data demonstrate that both the plp and golli genes are transcribed in the fetal human thymus. Moreover, both the PLP and DM-20 transcripts are produced from the plp gene, and the HOG 7 and HOG 5 transcripts are produced from the golli gene. Confocal fluorescent immunohistochemistry using antibodies for the PLP/DM-20 and Golli proteins, co-localized expression of these antigens to thymic macrophages. Thus, the plp and golli genes are expressed, and their corresponding protein produced, in an antigen presenting cell in the human immune system.
Assuntos
Regulação da Expressão Gênica/fisiologia , Proteína Básica da Mielina/genética , Proteína Proteolipídica de Mielina/genética , Timo/metabolismo , Processamento Alternativo , Northern Blotting , Feto/metabolismo , Humanos , Imuno-Histoquímica , Macrófagos/metabolismo , Reação em Cadeia da Polimerase/métodos , Timo/embriologia , Transcrição GênicaRESUMO
We have analyzed human fetal thymus and spleen for expression of the proteolipid protein (PLP) gene. We demonstrate that the PLP gene is transcribed in both tissues, and that both the PLP and DM-20 mRNAs are produced. Western blot analyses revealed that both the PLP and DM-20 protein isoforms were present in the fetal thymus and spleen. Immunohistochemical analyses indicated that the PLP/DM-20 proteins were detected in cells which have the distribution and morphology of thymic macrophages. These results provide further evidence that the PLP and DM-20 proteins are expressed in cell types other than myelin forming cells and possess function(s) unrelated to myelin structure. Furthermore, these data demonstrate that the PLP and DM-20 proteins are not shielded from the immune system behind the blood-brain barrier. These observations directly impinge upon the debate concerning acquisition of tolerance and the recognition that the encephalitogenic nature of PLP in diseases, such as Multiple Sclerosis, may not simply be related to its 'sequestration' from a 'naive' immune system.
Assuntos
Feto/fisiologia , Proteína Proteolipídica de Mielina/genética , Timo/embriologia , Processamento Alternativo/fisiologia , Northern Blotting , Western Blotting , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Humanos , Imuno-Histoquímica , Proteína Proteolipídica de Mielina/análise , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Baço/química , Baço/fisiologia , Linfócitos T/química , Linfócitos T/imunologia , Timo/citologiaRESUMO
Vasoactive intestinal peptide (VIP) exhibits pronounced effects on the growth rate of cultured mouse embryonic day (E) 9.5 embryos and acts in tissue culture as a potent glial mitogen and neuron survival factor. However, previous studies using immunohistochemistry or in situ hybridization in the rat have not revealed the presence and location of VIP or VIP mRNA in the early developing embryo CNS. Using a sensitive in situ hybridization assay with a 33P-labeled riboprobe, we show here that the VIP gene is expressed at least as early as E11 in the mouse hindbrain. Northern blot analysis on RNA from brain dissected from mouse embryos beginning at E14 confirmed that a correct-size mRNA for VIP was present by E14 and at later time points. Expression of the VIP2 receptor gene was also detected by northern analysis in E14 mouse brains. These studies support the hypothesis that VIP produced by the embryo exerts important effects on embryonic nervous system development.
Assuntos
Desenvolvimento Embrionário e Fetal/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Receptores de Peptídeo Intestinal Vasoativo/genética , Peptídeo Intestinal Vasoativo/genética , Animais , Northern Blotting , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Hibridização In Situ , Camundongos , Camundongos Endogâmicos BALB C , Gravidez , RNA Mensageiro/análiseRESUMO
A cDNA was isolated from a zebra finch telencephalon cDNA library that encodes the myelin proteolipid protein. The clone was 2874 nucleotides long containing an open reading frame of 831 nucleotides that encoded a 277 amino acid myelin proteolipid protein. The 5'- and 3' untranslated regions were 112 and 1931 nucleotides, respectively. In Northern blots the clone hybridized to 3 bands of 3.5, 2.4 and 1.5 Kb in mouse brain RNA, but to only a single band of 3.0 kb in zebra finch brain RNA, suggesting the lack of alternative polyadenylation sites within the 3' untranslated region of the zebra finch PLP mRNAs. There was a small degree of homology between the zebra finch and chicken PLP 5' untranslated regions, but relatively little homology of the 5' untranslated regions of the zebra finch PLP cDNA clone with the homologous regions of PLP cDNAs of many mammalian species. Except for a small stretch of considerable homology, there was little overall homology with the 3' untranslated regions of mammalian PLP mRNAs. Approximately 10% (i.e. 28) of the amino acids in the zebra finch PLP differed from mammalian PLP, with most of these changes located within exon 3. There were 16 amino acid changes between zebra finch and chicken, suggesting that greater sequence variation in PLP structure is tolerated among avian species than among mammalian species.
Assuntos
Aves/genética , DNA Complementar/isolamento & purificação , Proteínas da Mielina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Código Genético , Dados de Sequência Molecular , Proteína Proteolipídica de Mielina , RNA Mensageiro/genética , Homologia de Sequência do Ácido NucleicoRESUMO
The myelin basic protein (MBP) gene expresses several alternatively spliced products with the same 5' and 3' untranslated regions (UTRs). It has been reported that its expression may be regulated not only at the transcriptional level but also at the translational level during development. We engineered several MBP mRNA deletion mutants with 5' (-48, -37, -27, -22, and -10) and 3' UTRs of differing lengths and examined the translational efficiencies of these constructs in cell-free systems. The translational efficiencies of the constructs differed significantly over a range of almost 10-fold. A deletion of 11 nucleotides from the 5' end of the natural (i.e., -48) MBP mRNA resulted in an approximate fourfold reduction in translational efficiency. Further truncation of the 5' UTR increased the translational efficiencies of the constructs as has been observed with many RNAs. These results suggest that there may be a positive control element between -48 and -37 nucleotides in the 5' UTR of MBP mRNA. The effects of modifying the lengths of the 5' UTR on the translational efficiency of mRNAs encoding the 21.5-kDa and 14-kDa MBPs were the same, suggesting that the effect observed was not unique to the 21.5-kDa MBP mRNA. Truncating the 3' UTR of four different alternatively spliced MBP mRNAs also altered their translational efficiencies. Thus, the 5' and 3' UTRs of MBP mRNAs appear to influence the translation of these mRNAs, and such factors may be involved in the translational regulation of MBP gene expression.
Assuntos
Proteína Básica da Mielina/genética , Biossíntese de Proteínas , RNA Mensageiro/química , Animais , Sequência de Bases , Sistema Livre de Células , Deleção de Genes , Expressão Gênica , Dados de Sequência Molecular , Mutagênese , Reação em Cadeia da Polimerase , Splicing de RNA , RNA Mensageiro/genética , Coelhos , Reticulócitos/metabolismo , TriticumRESUMO
Two human Golli (for gene expressed in the oligodendrocyte lineage)-MBP (for myelin basic protein) cDNAs have been isolated from a human oligodendroglioma cell line. Analysis of these cDNAs has enabled us to determine the entire structure of the human Golli-MBP gene. The Golli-MBP gene, which encompasses the MBP transcription unit, is approximately 179 kb in length and consists of 10 exons, seven of which constitute the MBP gene. The human Golli-MBP gene contains two transcription start sites, each of which gives rise to a family of alternatively spliced transcripts. At least two Golli-MBP transcripts, containing the first three exons of the gene and one or more MBP exons, are produced from the first transcription start site. The second family of transcripts contains only MBP exons and produces the well-known MBPs. In humans, RNA blot analysis revealed that Golli-MBP transcripts were expressed in fetal thymus, spleen, and human B-cell and macrophage cell lines, as well as in fetal spinal cord. These findings clearly link the expression of exons encoding the autoimmunogen/encephalitogen MBP in the central nervous system to cells and tissues of the immune system through normal expression of the Golli-MBP gene. They also establish that this genetic locus, which includes the MBP gene, is conserved among species, providing further evidence that the MBP transcription unit is an integral part of the Golli transcription unit and suggest that this structural arrangement is important for the genetic function and/or regulation of these genes.
Assuntos
Proteína Básica da Mielina/genética , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Éxons , Genes , Humanos , Camundongos , Dados de Sequência Molecular , Oligodendroglioma/genética , RNA Neoplásico/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , Transcrição GênicaRESUMO
We have identified a novel transcription unit of 105 kilobases (called the Golli-mbp gene) that encompasses the mouse myelin basic protein (MBP) gene. Three unique exons within this gene are alternatively spliced into MBP exons and introns to produce a family of MBP gene-related mRNAs that are under individual developmental regulation. These mRNAs are temporally expressed within cells of the oligodendrocyte lineage at progressive stages of differentiation. Thus, the MBP gene is a part of a more complex gene structure, the products of which may play a role in oligodendrocyte differentiation prior to myelination. One Golli-mbp mRNA that encodes a protein antigenically related to MBP is also expressed in the spleen and other non-neural tissues.
Assuntos
Encéfalo/metabolismo , Proteína Básica da Mielina/genética , Oligodendroglia/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/citologia , Encéfalo/embriologia , Células Cultivadas , DNA , Regulação da Expressão Gênica , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , RNA Mensageiro/metabolismoRESUMO
We have conditionally immortalized oligodendrocytes isolated from normal and shiverer primary mouse brain cultures through the use of the retroviral vector ZIPSVtsA58. This vector encodes an immortalizing thermolabile simian virus 40 large T antigen (Tag) and allows for clonal selection by conferring neomycin (G418) resistance. We isolated 14 shiverer and 10 normal lines that expressed the early oligodendrocyte marker 2',3'-cyclic nucleotide 3'-phosphodiesterase mRNA. These cell lines grew continuously at the permissive temperature (34 degrees C) and displayed Tag nuclear immunostaining. On shifting to nonpermissive temperatures (39 degrees C), the cells showed rapid arrested cell growth and loss of Tag staining. One line (N20.1) engineered from normal oligodendrocytes also expressed myelin basic protein (MBP) and proteolipid protein (PLP) mRNAs, genes normally expressed by mature, differentiated oligodendrocytes. No differences in any of the myelin-specific protein mRNA levels were observed in N20.1 cells grown at 39 degrees C for > 9 days compared with cells maintained at 34 degrees C. Immunocytochemical staining revealed N20.1 cells to be positive for the oligodendrocyte surface markers--galactocerebroside, A007, and A2B5. However, MBP and PLP polypeptides could not be detected by western blot or immunocytochemical staining at either the permissive or nonpermissive temperature. Cell-free protein synthesis experiments indicated that the MBP mRNAs isolated from N20.1 cells were translatable and directed the synthesis of the 17-, 18.5-, and 21.5-kDa MBP isoforms. Analysis of the PLP/DM20 gene splice products by polymerase chain reaction indicated that the expression of DM20 mRNA predominated over that of PLP mRNA in this cell line. Because the cell line expressed the MBP and PLP genes, it represents a "mature" oligodendrocyte, but the splicing patterns of these genes indicate that it is at an early stage of "maturation." This cell line has now been passaged > 40 times with fidelity of phenotype and genotype.
Assuntos
Encéfalo/metabolismo , Transformação Celular Viral , Proteínas da Mielina/genética , Bainha de Mielina/fisiologia , Oligodendroglia/metabolismo , RNA Mensageiro/metabolismo , Vírus 40 dos Símios/genética , 2',3'-Nucleotídeo Cíclico Fosfodiesterases/genética , 2',3'-Nucleotídeo Cíclico Fosfodiesterases/metabolismo , Animais , Animais Recém-Nascidos , Antígenos Transformantes de Poliomavirus/genética , Sequência de Bases , Linhagem Celular Transformada , Células Cultivadas , Cruzamentos Genéticos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Mutantes Neurológicos , Dados de Sequência Molecular , Proteína Básica da Mielina/genética , Oligodesoxirribonucleotídeos , Reação em Cadeia da Polimerase/métodos , Biossíntese de Proteínas , Proteolipídeos/genética , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , TemperaturaRESUMO
Northern blot analysis of poly A(+) RNA isolated from mouse heart revealed the expression of 3.3 and 2.4 kb mRNAs that hybridized with a cDNA for the mouse proteolipid protein (PLP). In order to examine the relationship of these RNAs to the myelin PLP/DM20 mRNAs, a mouse heart cDNA library was prepared and screened with a mouse PLP cDNA. A cDNA was isolated, sequenced, and found to encode the DM20 variant of PLP. Polymerase chain reaction (PCR) analysis of heart cDNA with three sets of primers confirmed the presence of DM20 mRNA in mouse heart and indicated that it is the major splice product of the PLP gene expressed in that tissue. In situ hybridization localized the expression of the DM20 mRNA to the myocardial cells. Northern blot analysis indicated that expression of the DM20 mRNA is developmentally regulated in the murine heart, increasing significantly in concentration after 12 days postpartum. Northern analysis also revealed the expression of the DM20 mRNA in the hearts of the jimpy and quaking mutants. These results indicate that the PLP gene is expressed in tissues other than brain and support the concept that products of the PLP gene may have some biological role other than as structural components of myelin.