RESUMO
The propagation of X chromosome inactivation is thought to be mediated by the cis- limited spreading of the non-protein coding Xist transcript. In this report we have investigated the localization of Xist RNA on rodent metaphase chromosomes. We show that Xist RNA exhibits a banded pattern on the inactive X and is excluded from regions of constitutive heterochromatin. The banding pattern suggests a preferential association with gene-rich, G-light regions. Analysis of X:autosome rearrangements revealed that restricted propagation of X inactivation into cis -linked autosomal material is reflected by a corresponding limited spread of Xist RNA. We discuss these results in the context of models for the function of Xist RNA in the propagation of X inactivation.
Assuntos
Mecanismo Genético de Compensação de Dose , RNA não Traduzido , Fatores de Transcrição/genética , Cromossomo X/genética , Animais , Arvicolinae , Células Cultivadas , Mapeamento Cromossômico , Feminino , Hibridização in Situ Fluorescente , Masculino , Metáfase/genética , Camundongos , RNA/genética , RNA Longo não Codificante , Translocação GenéticaRESUMO
Developmental regulation of the mouse Xist gene at the onset of X chromosome inactivation is mediated by RNA stabilization. Here, we show that alternate promoter usage gives rise to distinct stable and unstable RNA isoforms. Unstable Xist transcript initiates at a novel upstream promoter, whereas stable Xist RNA is transcribed from the previously identified promoter and from a novel downstream promoter. Analysis of cells undergoing X inactivation indicates that a developmentally regulated promoter switch mediates stabilization and accumulation of Xist RNA on the inactive X chromosome.
Assuntos
Mecanismo Genético de Compensação de Dose , Regulação da Expressão Gênica no Desenvolvimento , Regiões Promotoras Genéticas/fisiologia , RNA não Traduzido , Fatores de Transcrição/genética , Cromossomo X , Animais , Linhagem Celular , Desenvolvimento Embrionário e Fetal/genética , Hibridização in Situ Fluorescente , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , RNA Longo não Codificante , RNA Mensageiro/metabolismo , Transcrição Gênica/genéticaRESUMO
Comparative mapping of X-linked genes has progressed rapidly since Ohno's prediction that genes on the X chromosome should be conserved as a syntenic group in all mammals. Although several conserved blocks of homology between human and mouse have been discovered, rearrangements within the X chromosome have also been characterized. More recently, some exceptions to Ohno's law have been reported. We have used fluorescence in situ hybridization (FISH) to map five genes, Gla, G6pd, Hprt, Pgk1 and Xist, to two of the largest conserved segments of X material in five members of the genus Microtus (grey vole) and show that vole X chromosomes demonstrate greater homology to human than to mouse. Cytogenetic analysis indicates a relatively high frequency of rearrangement during vole evolution, although certain blocks of homology appear to be highly conserved in all species studied to date. On this basis we were able to predict the probable location of the rat X inactivation centre (Xic) based solely on high-resolution G-banding. Our prediction was then confirmed by mapping the rat Xist gene by FISH. The possible significance of conserving long-range chromosome structure in the vicinity of the Xic is discussed with respect to the mechanism of X inactivation.
Assuntos
Arvicolinae/genética , Mapeamento Cromossômico , RNA não Traduzido , Cromossomo X/genética , Animais , Células Cultivadas , Sondas de DNA , Mecanismo Genético de Compensação de Dose , Evolução Molecular , Fibroblastos , Rearranjo Gênico , Ligação Genética , Glucosefosfato Desidrogenase/genética , Hipoxantina Fosforribosiltransferase/genética , Hibridização in Situ Fluorescente/métodos , Masculino , Fosfoglicerato Quinase/genética , RNA Longo não Codificante , Ratos , Homologia de Sequência do Ácido Nucleico , Fatores de Transcrição/genética , alfa-Galactosidase/genéticaRESUMO
Recent years have seen rapid progress towards understanding the molecular mechanisms involved in X chromosome inactivation (X inactivation). This progress has largely revolved around the discovery of the X inactive specific transcript (Xist) gene, which is known now to represent the master switch locus regulating X inactivation. In adult cells Xist is transcribed exclusively from the inactive X chromosome. The transcript has no apparent protein-coding potential and is retained in the nucleus in close association with the domain occupied by the inactive X chromosome. It is thus thought to represent a functional RNA molecule which acts as the primary signal responsible for the propagation of X inactivation. Developmental regulation of Xist correlates with the developmental timing of X inactivation. Recent results have demonstrated that Xist is both necessary and sufficient for X inactivation. Goals for the future are to understand the mechanism of Xist regulation which underlies the establishment of appropriate X inactivation patterns and to determine how Xist RNA participates in the process of propagating inactivation in cis.
Assuntos
Mecanismo Genético de Compensação de Dose , RNA não Traduzido , Fatores de Transcrição/genética , Cromossomo X/genética , Animais , Fixação Psicológica Instintiva , RNA Longo não CodificanteRESUMO
We have characterized two novel, complex, heterochromatic repeat sequences, MS3 and MS4, isolated from Microtus rossiaemeridionalis genomic DNA. Sequence analysis indicates that both repeats consist of unique sequences interrupted by repeat elements of different origin and can be classified as long complex repeat units (LCRUs). A unique feature of both repeat units is the presence of short interspersed repeat elements (SINEs), which are usually characteristic of the euchromatic part of the genome. Comparative analysis revealed no significant stretches of homology in the nucleotide sequences between the two repeats, suggesting that the repeats originated independently during the course of vole genome evolution. Fluorescence in situ hybridization analysis demonstrates that MS3 and MS4 occupy distinct domains in the heterochromatic regions of the sex chromosomes in M. transcaspicus and M. arvalis but collocalize in M. rossiaemeridionalis and M. kirgisorum heterochromatic blocks. The localization pattern of the repeats on the vole chromosomes confirms the independent origin of the two repeats and suggests that expansion of the heterochromatic blocks has occurred subsequent to speciation.
Assuntos
Arvicolinae/genética , Mapeamento Cromossômico , Sequências Repetitivas de Ácido Nucleico , Animais , Sequência de Bases , Southern Blotting , Clonagem Molecular , Citogenética , Evolução Molecular , Feminino , Heterocromatina , Hibridização in Situ Fluorescente , Masculino , Camundongos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Alinhamento de Sequência , Análise de Sequência de DNA , Cromossomos SexuaisRESUMO
The onset of X inactivation is preceded by a marked increase in the level of Xist RNA. Here we demonstrate that increased stability of Xist RNA is the primary determinant of developmental up-regulation. Unstable transcript is produced by both alleles in XX ES cells and in XX embryos prior to the onset of random X inactivation. Following differentiation, transcription of unstable RNA from the active X chromosome allele continues for a period following stabilization and accumulation of transcript on the inactive X allele. We discuss the implications of these findings in terms of models for the initiation of random and imprinted X inactivation.
Assuntos
Mecanismo Genético de Compensação de Dose , RNA Mensageiro/metabolismo , RNA não Traduzido , Fatores de Transcrição/genética , Alelos , Animais , Blastocisto , Diferenciação Celular , Células Cultivadas , Dactinomicina/farmacologia , Feminino , Regulação da Expressão Gênica/fisiologia , Masculino , Camundongos , Modelos Genéticos , Inibidores da Síntese de Ácido Nucleico/farmacologia , RNA Longo não Codificante , RNA Mensageiro/biossíntese , Células-Tronco , Transcrição Gênica/fisiologiaAssuntos
Encéfalo/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas do Tecido Nervoso/genética , Sequência de Aminoácidos , Encéfalo/embriologia , Proteínas de Ligação a DNA/biossíntese , Proteínas de Grupo de Alta Mobilidade/biossíntese , Humanos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/biossíntese , Fases de Leitura Aberta , Fatores de Transcrição SOXB1 , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
A cDNA for human thyrotropin-releasing hormone (TRH) receptor has been isolated from a human pituitary cDNA library. By using this cDNA as a biotinylated probe, the gene encoding the TRH receptor has been localized to chromosome 8q23 by in situ hybridization.
Assuntos
Cromossomos Humanos Par 8 , Receptores do Hormônio Liberador da Tireotropina/genética , Mapeamento Cromossômico , Sondas de DNA , DNA Complementar , Humanos , Hibridização in Situ Fluorescente , Dados de Sequência MolecularRESUMO
The cloning and characterization of the mouse TRH receptor (TRH-R) gene revealed an untranslated exon (exon 1), a single intron and an upstream dinucleotide repeat sequence (d(TG)16.d(AG)21) in the 5' untranslated region (UTR). The coding region was contained almost entirely on a second exon (exon 2), with the final amino acid and stop codon at the COOH terminus of the gene encoded by a third exon (exon 3) flanked by two introns. The 3' UTR was contained on the remainder of exon 3 and on the final exon (exon 4). Exon 3 (228 bp) corresponds exactly to a 228 bp deletion that exists in the rat TRH-R cDNA, but not in the mouse cDNA. The mouse TRH-R cDNA encodes a protein of 393 amino acids which is 96% homologous to the rat TRH-R protein of 412 amino acids, but is 19 amino acids shorter at its COOH terminus. The coding sequence for these 19 amino acids (plus 1 extra amino acid) does exist in the mouse TRH-R gene, but the sequence is encoded by exon 4, separated from the rest of the coding region by the stop codon and 223 bp of 3' UTR on exon 3. Splicing of exon 3 in the mouse TRH-R gene would remove the last amino acid, the stop codon and the 223 bp of 3' UTR, allowing transcription to continue into the 3' UTR on exon 4, which encodes the 19 extra amino acids found in the rat cDNA.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Genes , Camundongos/genética , Receptores do Hormônio Liberador da Tireotropina/genética , Deleção de Sequência , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar/genética , Éxons , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Ratos/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Especificidade da EspécieRESUMO
Thyrotrophin-releasing hormone (TRH) is a hypothalamic tripeptide known to act via its receptor in the anterior pituitary gland to stimulate the release of thyrotrophin (TSH) from thyrotrophs and prolactin (PRL) from lactotrophs. It is also thought to act as a neurotransmitter/neuromodulator in the central and peripheral nervous systems. We have isolated a cDNA encoding the human pituitary TRH receptor (TRH-R) protein with a predicted amino acid sequence of 398 amino acids. Comparison of the amino acid sequences of the human TRH-R with the previously published rodent TRH-Rs showed that it is similar to both the rat and mouse TRH-Rs, except that each species has variant amino acids at the carboxy (COOH) terminus. The human TRH-R belongs to the family of seven transmembrane domain, G-protein-coupled receptors, and it is believed that the COOH terminal region of this family of receptors may play an important role in receptor downregulation/internalisation, and possibly G-protein coupling. COS-1 cells expressing the human TRH-R showed high affinity receptor binding. Stimulation of these cells with TRH produced a typical phosphoinositide response and mobilisation of intracellular calcium [Ca2+]i.