RESUMEN
The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus is thought to have originated in wild bats from Asia, and as the resulting pandemic continues into its third year, concerns have been raised that the virus will expand its host range and infect North American wildlife species, including bats. Mexican free-tailed bats (Tadarida brasiliensis) live in large colonies in the southern United States, often in urban areas and, as such, could be exposed to the virus from infected humans. We experimentally challenged wild T. brasiliensis with SARS-CoV-2 to determine the susceptibility, reservoir potential, and population impacts of infection in this species. Of 10 bats oronasally inoculated with SARS-CoV-2, 5 became infected and orally excreted moderate amounts of virus for up to 18 days postinoculation. These five subjects all seroconverted and cleared the virus before the end of the study with no obvious clinical signs of disease. We additionally found no evidence of viral transmission to uninoculated subjects. These results indicate that while T. brasiliensis are susceptible to SARS-CoV-2 infection, infection of wild populations of T. brasiliensis would not likely cause mortality. However, the transmission of SARS-CoV-2 from T. brasiliensis to or from humans, or to other animal species, is a possibility requiring further investigation to better define. IMPORTANCE As the COVID-19 pandemic has continued for 3+ years, there has been increasing concern that the SARS-CoV-2 virus will enter wildlife populations and potentially create new reservoirs where the virus could adapt to a new host and create variants. This is particularly possible with species that reside in man-made structures, in proximity to infected human populations. Mexican free-tailed bats (Tadarida brasiliensis) live in large colonies, often in urban settings and, thus, can be exposed by infected humans and potentially transmit the virus to new hosts. We experimentally challenged T. brasiliensis with SARS-CoV-2 and revealed that they are susceptible to the virus and excrete moderate amounts for up to 18 days postinoculation. This is important information for wildlife biologists, wildlife rehabilitation workers, and the general public that may contact these animals.
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COVID-19 , Quirópteros , Animales , Humanos , SARS-CoV-2 , Pandemias , Animales SalvajesRESUMEN
The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus originated in wild bats from Asia, and as the resulting pandemic continues into its third year, concerns have been raised that the virus will expand its host range and infect North American wildlife species, including bats. Mexican free-tailed bats ( Tadarida brasiliensis : TABR) live in large colonies in the southern United States, often in urban areas, and as such, could be exposed to the virus from infected humans. We experimentally challenged wild TABR with SARS-CoV-2 to determine the susceptibility, reservoir potential, and population impacts of infection in this species. Of nine bats oronasally inoculated with SARS-CoV-2, five became infected and orally excreted moderate amounts of virus for up to 18 days post inoculation. These five subjects all seroconverted and cleared the virus before the end of the study with no obvious clinical signs of disease. We additionally found no evidence of viral transmission to uninoculated subjects. These results indicate that while TABR are susceptible to SARS-CoV-2 infection, infection of wild populations of TABR would not likely cause mortality. However, the transmission of SARS-CoV-2 from TABR to or from humans, or to other animal species, is a distinct possibility requiring further investigation to better define.
RESUMEN
Pigeon paramyxovirus serotype 1 (PPMV-1) is a globally distributed, virulent member of the avian paramyxovirus serotype 1 serogroup that causes mortality in columbiformes and poultry. Following introduction into the United States in the mid-1980s, PPMV-1 rapidly spread causing numerous mortality events in Eurasian collared-doves ( Streptopelia decaocto) (ECDOs) and rock pigeons ( Columba livia) (ROPIs). The investigators reviewed pathological findings of 70 naturally infected, free-ranging columbiforms from 25 different mortality events in the United States. Immunohistochemistry targeting PPMV-1 nucleoprotein was used to determine the tissue distribution of the virus in a subset of 17 birds from 10 of the studied outbreaks. ECDOs (61 birds) and ROPIs (9 birds) were the only species in which PPMV-1-associated disease was confirmed by viral isolation and presence of histologic lesions. Acute to subacute tubulointerstitial nephritis and necrotizing pancreatitis were the most frequent histologic lesions, with immunolabeling of viral antigen in renal tubular epithelial cells and pancreatic acinar epithelium. Lymphoid depletion of bursa of Fabricius and spleen was common, but the presence of viral antigen in these organs was inconsistent among infected birds. Hepatocellular necrosis was occasionally present with immunolabeling of hypertrophic Kupffer cells, and immunopositive eosinophilic intracytoplasmic inclusion bodies were present in hepatocytes of 1 ECDO. Immunopositive lymphocytic choroiditis was present in 1 ECDO, while lymphocytic meningoencephalitis was frequent in ROPIs in absence of immunolabeling. This study demonstrates widespread presence of PPMV-1 antigen in association with histologic lesions, confirming the lethal potential of this virus in these particular bird species.
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Columbidae/virología , Enfermedad de Newcastle/virología , Virus de la Enfermedad de Newcastle , Animales , Animales Salvajes/virología , Bolsa de Fabricio/patología , Bolsa de Fabricio/virología , Brotes de Enfermedades/veterinaria , Nefritis Intersticial/patología , Nefritis Intersticial/veterinaria , Nefritis Intersticial/virología , Enfermedad de Newcastle/epidemiología , Enfermedad de Newcastle/patología , Virus de la Enfermedad de Newcastle/aislamiento & purificación , Bazo/patología , Bazo/virología , Estados Unidos/epidemiologíaRESUMEN
A novel highly pathogenic avian influenza virus belonging to the H5 clade 2.3.4.4 variant viruses was detected in North America in late 2014. Motivated by the identification of these viruses in domestic poultry in Canada, an intensive study was initiated to conduct highly pathogenic avian influenza surveillance in wild birds in the Pacific Flyway of the United States. A total of 4,729 hunter-harvested wild birds were sampled and highly pathogenic avian influenza virus was detected in 1.3% (n = 63). Three H5 clade 2.3.4.4 subtypes were isolated from wild birds, H5N2, H5N8, and H5N1, representing the wholly Eurasian lineage H5N8 and two novel reassortant viruses. Testing of 150 additional wild birds during avian morbidity and mortality investigations in Washington yielded 10 (6.7%) additional highly pathogenic avian influenza isolates (H5N8 = 3 and H5N2 = 7). The geographically widespread detection of these viruses in apparently healthy wild waterfowl suggest that the H5 clade 2.3.4.4 variant viruses may behave similarly in this taxonomic group whereby many waterfowl species are susceptible to infection but do not demonstrate obvious clinical disease. Despite these findings in wild waterfowl, mortality has been documented for some wild bird species and losses in US domestic poultry during the first half of 2015 were unprecedented.
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Aves/virología , Subtipo H5N1 del Virus de la Influenza A/aislamiento & purificación , Subtipo H5N2 del Virus de la Influenza A/aislamiento & purificación , Animales , Animales Salvajes/virología , Canadá , Brotes de Enfermedades , Gripe Aviar/virología , América del Norte , Aves de Corral/virología , Enfermedades de las Aves de Corral/virología , Virus Reordenados/aislamiento & purificación , Estados UnidosRESUMEN
UNLABELLED: Describing the viral diversity of wildlife can provide interesting and useful insights into the natural history of established human pathogens. In this study, we describe a previously unknown picornavirus in harbor seals (tentatively named phopivirus) that is related to human hepatitis A virus (HAV). We show that phopivirus shares several genetic and phenotypic characteristics with HAV, including phylogenetic relatedness across the genome, a specific and seemingly quiescent tropism for hepatocytes, structural conservation in a key functional region of the type III internal ribosomal entry site (IRES), and a codon usage bias consistent with that of HAV. IMPORTANCE: Hepatitis A virus (HAV) is an important viral hepatitis in humans because of the substantial number of cases each year in regions with low socioeconomic status. The origin of HAV is unknown, and no nonprimate HAV-like viruses have been described. Here, we describe the discovery of an HAV-like virus in seals. This finding suggests that the diversity and evolutionary history of these viruses might be far greater than previously thought and may provide insight into the origin and pathogenicity of HAV.
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Hepatovirus/genética , Hepatovirus/aislamiento & purificación , Filogenia , Phocidae/virología , Animales , Codón , Genoma Viral , Genotipo , Virus de la Hepatitis A Humana/genética , Hepatovirus/fisiología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Hígado/virología , Pulmón/virología , ARN Viral/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Bazo/virología , Replicación ViralRESUMEN
UNLABELLED: From September to December 2011, 162 New England harbor seals died in an outbreak of pneumonia. Sequence analysis of postmortem samples revealed the presence of an avian H3N8 influenza A virus, similar to a virus circulating in North American waterfowl since at least 2002 but with mutations that indicate recent adaption to mammalian hosts. These include a D701N mutation in the viral PB2 protein, previously reported in highly pathogenic H5N1 avian influenza viruses infecting people. Lectin staining and agglutination assays indicated the presence of the avian-preferred SAα-2,3 and mammalian SAα-2,6 receptors in seal respiratory tract, and the ability of the virus to agglutinate erythrocytes bearing either the SAα-2,3 or the SAα-2,6 receptor. The emergence of this A/harbor seal/Massachusetts/1/2011 virus may herald the appearance of an H3N8 influenza clade with potential for persistence and cross-species transmission. IMPORTANCE: The emergence of new strains of influenza virus is always of great public concern, especially when the infection of a new mammalian host has the potential to result in a widespread outbreak of disease. Here we report the emergence of an avian influenza virus (H3N8) in New England harbor seals which caused an outbreak of pneumonia and contributed to a U.S. federally recognized unusual mortality event (UME). This outbreak is particularly significant, not only because of the disease it caused in seals but also because the virus has naturally acquired mutations that are known to increase transmissibility and virulence in mammals. Monitoring the spillover and adaptation of avian viruses in mammalian species is critically important if we are to understand the factors that lead to both epizootic and zoonotic emergence.
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Enfermedades Transmisibles Emergentes/veterinaria , Subtipo H3N8 del Virus de la Influenza A/aislamiento & purificación , Infecciones por Orthomyxoviridae/veterinaria , Phoca/virología , Neumonía/veterinaria , Animales , Enfermedades Transmisibles Emergentes/epidemiología , Enfermedades Transmisibles Emergentes/virología , Brotes de Enfermedades , Humanos , Subtipo H3N8 del Virus de la Influenza A/clasificación , Subtipo H3N8 del Virus de la Influenza A/genética , Subtipo H3N8 del Virus de la Influenza A/patogenicidad , Subtipo H5N1 del Virus de la Influenza A/genética , Subtipo H5N1 del Virus de la Influenza A/aislamiento & purificación , Gripe Humana/virología , Datos de Secuencia Molecular , Mutación , New England/epidemiología , Infecciones por Orthomyxoviridae/epidemiología , Infecciones por Orthomyxoviridae/virología , Filogenia , Neumonía/epidemiología , Neumonía/virología , Proteínas Virales/genética , VirulenciaRESUMEN
A method to quantify sub-ppm levels of formaldehyde in acetone has been developed and it is reported here. In this method, the different reactivities and stabilities of sulfite with formaldehyde and acetone are used to separate the two carbonyl compounds. Sulfite reacts with formaldehyde to form hydroxymethanesulfonate (HMS), the non-volatile and stable nature of which allows its separation from bulk acetone solvent. The resulting HMS is then converted back to formaldehyde under basic conditions, and formaldehyde is derivatized with 2,4-dinitrophenylhydrazine (DNPH) and quantified in its DNP hydrazone form using high-performance liquid chromatography-UV detection. The method detection limit at the 99% confidence level was 0.051 mg L(-1). A batch of samples can be processed within 4 h. The method has been applied to quantify the amount of formaldehyde in an analytical-grade acetone and in a commercial nail polish remover and the level of formaldehyde was found to be 0.175 and 0.184 mg L(-1), respectively.
RESUMEN
RNA quantitation is becoming increasingly important in basic, pharmaceutical, and clinical research. For example, quantitation of viral RNAs can predict disease progression and therapeutic efficacy. Likewise, gene expression analysis of diseased versus normal, or untreated versus treated, tissue can identify relevant biological responses or assess the effects of pharmacological agents. As the focus of the Human Genome Project moves toward gene expression analysis, the field will require a flexible RNA analysis technology that can quantitatively monitor multiple forms of alternatively transcribed and/or processed RNAs (refs 3,4). We have applied the principles of invasive cleavage and engineered an improved 5'-nuclease to develop an isothermal, fluorescence resonance energy transfer (FRET)-based signal amplification method for detecting RNA in both total RNA and cell lysate samples. This detection format, termed the RNA invasive cleavage assay, obviates the need for target amplification or additional enzymatic signal enhancement. In this report, we describe the assay and present data demonstrating its capabilities for sensitive (<100 copies per reaction), specific (discrimination of 95% homologous sequences, 1 in > or =20,000), and quantitative (1.2-fold changes in RNA levels) detection of unamplified RNA in both single- and biplex-reaction formats.
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ARN/análisis , Espectrometría de Fluorescencia/métodos , Secuencia de Bases , Biotecnología/métodos , VIH/metabolismo , Modelos Genéticos , Datos de Secuencia Molecular , ARN/metabolismo , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Ácido NucleicoRESUMEN
A rapid and simple method for determining accessible sites in RNA that is independent of the length of target RNA and does not require RNA labeling is described. In this method, target RNA is allowed to hybridize with sequence-randomized libraries of DNA oligonucleotides linked to a common tag sequence at their 5'-end. Annealed oligonucleotides are extended with reverse transcriptase and the extended products are then amplified by using PCR with a primer corresponding to the tag sequence and a second primer specific to the target RNA sequence. We used the combination of both the lengths of the RT-PCR products and the location of the binding site of the RNA-specific primer to determine which regions of the RNA molecules were RNA extendible sites, that is, sites available for oligonucleotide binding and extension. We then employed this reverse transcription with the random oligonucleotide libraries (RT-ROL) method to determine the accessible sites on four mRNA targets, human activated ras (ha-ras), human intercellular adhesion molecule-1 (ICAM-1), rabbit beta-globin, and human interferon-gamma (IFN-gamma). Our results were concordant with those of other researchers who had used RNase H cleavage or hybridization with arrays of oligonucleotides to identify accessible sites on some of these targets. Further, we found good correlation between sites when we compared the location of extendible sites identified by RT-ROL with hybridization sites of effective antisense oligonucleotides on ICAM-1 mRNA in antisense inhibition studies. Finally, we discuss the relationship between RNA extendible sites and RNA accessibility.
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Globinas/genética , Molécula 1 de Adhesión Intercelular/genética , Interferón gamma/genética , Mapeo Nucleótido , ARN/genética , Proteínas ras/genética , Animales , Secuencia de Bases , Cartilla de ADN/química , Biblioteca de Genes , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Hibridación de Ácido Nucleico , Oligonucleótidos Antisentido/farmacología , Conejos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
GATA6 belongs to a family of zinc finger transcription factors that play important roles in transducing nuclear events that regulate cellular differentiation and embryonic morphogenesis in vertebrate species. To examine the function of GATA6 during embryonic development, gene targeting was used to generate GATA6-deficient (GATA6(-/-)) ES cells and mice harboring a null mutation in GATA6. Differentiated embryoid bodies derived from GATA6(-/-) ES cells lack a covering layer of visceral endoderm and severely attenuate, or fail to express, genes encoding early and late endodermal markers, including HNF4, GATA4, alpha-fetoprotein (AFP), and HNF3beta. Homozygous GATA6(-/-) mice died between embryonic day (E) 6.5 and E7. 5 and exhibited a specific defect in endoderm differentiation including severely down-regulated expression of GATA4 and absence of HNF4 gene expression. Moreover, widespread programmed cell death was observed within the embryonic ectoderm of GATA6-deficient embryos, a finding also observed in HNF4-deficient embryos. Consistent with these data, forced expression of GATA6 activated the HNF4 promoter in nonendodermal cells. Finally, to examine the function of GATA6 during later embryonic development, GATA6(-/-)-C57BL/6 chimeric mice were generated. lacZ-tagged GATA6(-/-) ES cells contributed to all embryonic tissues with the exception of the endodermally derived bronchial epithelium. Taken together, these data suggest a model in which GATA6 lies upstream of HNF4 in a transcriptional cascade that regulates differentiation of the visceral endoderm. In addition, these data demonstrate that GATA6 is required for establishment of the endodermally derived bronchial epithelium.
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Diferenciación Celular/genética , Proteínas de Unión al ADN/genética , Desarrollo Embrionario y Fetal/genética , Endodermo/citología , Regulación del Desarrollo de la Expresión Génica/genética , Fosfoproteínas/genética , Factores de Transcripción/genética , Vísceras/crecimiento & desarrollo , Células 3T3 , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Embrión de Mamíferos/patología , Factor de Transcripción GATA6 , Marcación de Gen , Genotipo , Factor Nuclear 4 del Hepatocito , Histocitoquímica , Hibridación in Situ , Pulmón/embriología , Ratones , Ratones Noqueados , Microscopía Electrónica , ARN Mensajero/genética , Activación Transcripcional/genéticaRESUMEN
The SM22alpha promoter has been used as a model system to define the molecular mechanisms that regulate smooth muscle cell (SMC) specific gene expression during mammalian development. The SM22alpha gene is expressed exclusively in vascular and visceral SMCs during postnatal development and is transiently expressed in the heart and somites during embryogenesis. Analysis of the SM22alpha promoter in transgenic mice revealed that 280 bp of 5' flanking sequence is sufficient to restrict expression of the lacZ reporter gene to arterial SMCs and the myotomal component of the somites. DNase I footprint and electrophoretic mobility shift analyses revealed that the SM22alpha promoter contains six nuclear protein binding sites (designated smooth muscle elements [SMEs] -1 to -6, respectively), two of which bind serum response factor (SRF) (SME-1 and SME-4). Mutational analyses demonstrated that a two-nucleotide substitution that selectively eliminates SRF binding to SME-4 decreases SM22alpha promoter activity in arterial SMCs by approximately 90%. Moreover, mutations that abolish binding of SRF to SME-1 and SME-4 or mutations that eliminate each SME-3 binding activity totally abolished SM22alpha promoter activity in the arterial SMCs and somites of transgenic mice. Finally, we have shown that a multimerized copy of SME-4 (bp -190 to -110) when linked to the minimal SM22alpha promoter (bp -90 to +41) is necessary and sufficient to direct high-level transcription in an SMC lineage-restricted fashion. Taken together, these data demonstrate that distinct transcriptional regulatory programs control SM22alpha gene expression in arterial versus visceral SMCs. Moreover, these data are consistent with a model in which combinatorial interactions between SRF and other transcription factors that bind to SME-4 (and that bind directly to SRF) activate transcription of the SM22alpha gene in arterial SMCs.
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Proteínas de Unión al ADN/metabolismo , Proteínas de Microfilamentos , Proteínas Musculares/genética , Músculo Liso/citología , Músculo Liso/metabolismo , Proteínas Nucleares/metabolismo , Animales , Secuencia de Bases , Sitios de Unión/genética , Células Cultivadas , ADN/genética , ADN/metabolismo , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Operón Lac , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Desarrollo de Músculos , Músculo Liso/crecimiento & desarrollo , Músculo Liso Vascular/citología , Músculo Liso Vascular/crecimiento & desarrollo , Músculo Liso Vascular/metabolismo , Mutación , Regiones Promotoras Genéticas , Ratas , Factor de Respuesta SéricaRESUMEN
Members of the GATA family of zinc finger transcription factors regulate critical steps of cellular differentiation during vertebrate development. In the studies described in this report, we have isolated and functionally characterized the murine GATA-5 cDNA and protein and defined the temporal and spatial pattern of GATA-5 gene expression during mammalian development. The amino terminus of the mouse GATA-5 protein shares high level amino acid sequence identity with the murine GATA-4 and -6 proteins, but not with other members of the GATA family. GATA-5 binds to the functionally important CEF-1 nuclear protein binding site in the cardiac-specific slow/cardiac troponin C (cTnC) transcriptional enhancer and overexpression of GATA-5 transactivates the cTnC enhancer in noncardiac muscle cell lines. During embryonic and postnatal development, the pattern of GATA-5 gene expression differs significantly from that of other GATA family members. In the primitive streak embryo, GATA-5 mRNA is detectable in the precardiac mesoderm. Within the embryonic heart, the GATA-5 gene is expressed within the atrial and ventricular chambers (ED 9.5), becomes restricted to the atrial endocardium (ED 12.5), and is subsequently not expressed in the heart during late fetal and postnatal development. Moreover, coincident with the earliest steps in lung development, only the GATA-5 gene is expressed within the pulmonary mesenchyme. Finally, the GATA-5 gene is expressed in tissue-restricted subsets of smooth muscle cells (SMCs), including bronchial SMCs and SMCs in the bladder wall. These data are consistent with a model in which GATA-5 performs a unique temporally and spatially restricted function in the embryonic heart and lung. Moreover, these data suggest that GATA-5 may play an important role in the transcriptional program(s) that underlies smooth muscle cell diversity.
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Proteínas de Unión al ADN/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Transactivadores/genética , Factores de Transcripción/genética , Células 3T3 , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células COS , Clonación Molecular , ADN Complementario , Desarrollo Embrionario y Fetal , Factor de Transcripción GATA5 , Gástrula/química , Corazón/embriología , Pulmón/embriología , Mesodermo/química , Ratones , Datos de Secuencia Molecular , Músculo Liso/química , Músculo Liso/citología , Especificidad de Órganos , ARN Mensajero/análisis , Homología de Secuencia de AminoácidoRESUMEN
GATA-4 is one of the earliest developmental markers of the precardiac mesoderm, heart, and gut and has been shown to activate regulatory elements controlling transcription of genes encoding cardiac-specific proteins. To elucidate the molecular mechanisms underlying the transcriptional activity of the GATA-4 protein, structure-function analyses were performed. These analyses revealed that the C-terminal zinc finger and adjacent basic domain of GATA-4 is bifunctional, modulating both DNA-binding and nuclear localization activities. The N terminus of the protein encodes two independent transcriptional Activation Domains (amino acids 1-74 and amino acids 130-177). Amino acid residues were identified within each domain that are required for transcriptional activation. Finally, we have shown that regions of Xenopus GATA-5 and -6 corresponding to Activation Domains I and II, respectively, function as potent transcriptional activators. The identification and functional characterization of two evolutionarily conserved transcriptional Activation Domains within the GATA-4/5/6 subfamily suggests that each of these domains modulates critical functions in the transcriptional regulatory program(s) encoded by GATA-4, -5, and -6 during vertebrate development. As such these data provide novel insights into the molecular mechanisms that control development of the heart.
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Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Transcripción Genética , Dedos de Zinc , Células 3T3 , Secuencia de Aminoácidos , Animales , Células COS , Secuencia Conservada , ADN/metabolismo , Factor de Transcripción GATA4 , Factor de Transcripción GATA5 , Factor de Transcripción GATA6 , Ratones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Alineación de Secuencia , Eliminación de Secuencia , Relación Estructura-Actividad , Troponina C/genéticaRESUMEN
The ELL gene was first identified by its involvement with MLL in the translocation (11;19)(q23;p13.1) in acute myeloid leukemia. To date, nine other MLL partner genes have been cloned, but their precise functions have yet to be determined. To characterize the functions of ELL further, we have cloned the murine homologue of ELL and have found that the gene is highly conserved at the nucleotide and amino acid level. The open reading frame of the murine homologue contains 602 aa, slightly smaller than the 621 aa in the human gene. With Northern blot analysis, a 3.4-kb transcript is detected in all tissues examined with greatest levels of expression in the liver. Unlike human ELL, only a single transcript can be detected with either murine coding sequence or 3' untranslated region probes. To examine the spatial and temporal pattern of expression in murine development, in situ hybridization studies were performed with sense and antisense riboprobes from the 3' untranslated region of murine Ell. Ell is expressed diffusely by embryonic day 7.5 (E7.5). In addition, high levels of expression can be detected in maternally derived decidual tissue. At E14.5, Ell is expressed diffusely throughout the embryo. However by E16.5, specific expression in the liver and gastrointestinal tract becomes prominent and remains so in both neonates and adults. To determine the subcellular localization of ELL, we developed a polyclonal antiserum to ELL that was used for immunofluorescence studies in COS-7, HeLa, NIH 3T3, and A7r5 cells. The ELL protein was localized to the nucleus but excluded from nucleoli in all cell lines examined. Recently, the gene product of ELL was found to function as an RNA polymerase II elongation factor, an activity that is consistent with our immunofluorescence data. Thus, these studies extend our understanding of the normal functions of ELL and provide additional insight into its aberrant function when fused to MLL in acute myeloid leukemia.
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Compartimento Celular , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de Neoplasias/aislamiento & purificación , Factores de Elongación de Péptidos , Factores de Transcripción/aislamiento & purificación , Enfermedad Aguda , Secuencia de Aminoácidos , Animales , Clonación Molecular , Secuencia Conservada , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos/química , Desarrollo Embrionario y Fetal , Técnica del Anticuerpo Fluorescente Indirecta , Hibridación in Situ , Leucemia Mieloide/etiología , Hígado/química , Ratones , Datos de Secuencia Molecular , Proteínas de Neoplasias/genética , ARN Polimerasa II/metabolismo , ARN Mensajero/análisis , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Factores de Elongación TranscripcionalRESUMEN
Members of the GATA family of zinc finger transcription factors play important roles in the development of several mesodermally derived cell lineages. In the studies described in this report, we have isolated and functionally characterized the murine GATA-6 cDNA and protein and defined the temporal and spatial patterns of GATA-6 gene expression during mammalian development. The GATA-6 and -4 proteins share high-level amino acid sequence identity over a proline-rich region at the amino terminus of the protein that is not conserved in other GATA family members. GATA-6 binds to a functionally important nuclear protein binding site within the cardiac-specific cardiac troponin C (cTnC) transcriptional enhancer. Moreover, the cTnC promoter enhancer can be transactivated by overexpression of GATA-6 in noncardiac muscle cells. During early murine embryonic development, the patterns of GATA-6 and -4 gene expression are similar, with expression of GATA-6 restricted to the precardiac mesoderm, the embryonic heart tube, and the primitive gut. However, coincident with the onset of vasculogenesis and development of the respiratory and urogenital tracts, only the GATA-6 gene is expressed in arterial smooth muscle cells, the developing bronchi, and the urogenital ridge and bladder. These data are consistent with a model in which GATA-6 functions in concert with GATA-4 to direct tissue-specific gene expression during formation of the mammalian heart and gastrointestinal tract, but performs a unique function in programming lineage-restricted gene expression in the arterial system, the bladder, and the embryonic lung.
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Proteínas de Unión al ADN/genética , Regulación del Desarrollo de la Expresión Génica , Mesodermo/fisiología , Factores de Transcripción/genética , Dedos de Zinc/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Sistema Cardiovascular/embriología , Linaje de la Célula/genética , Células Clonales , ADN Complementario/aislamiento & purificación , Sistema Digestivo/embriología , Factor de Transcripción GATA6 , Pulmón/embriología , Ratones , Datos de Secuencia Molecular , Músculo Esquelético/embriología , Músculo Liso Vascular/embriología , ARN/análisis , Ratas , Troponina/genética , Troponina C , Sistema Urogenital/embriologíaRESUMEN
Spi-B and PU.1 are hematopoietic-specific transcription factors that constitute a subfamily of the Ets family of DNA-binding proteins. Here we show that contrary to previous reports, PU.1 and Spi-B have very different expression patterns. PU.1 is expressed at high levels in B cells, mast cells, megakaryocytes, macrophages, neutrophils, and immature erythroid cells and at lower levels in mature erythrocytes. PU.1 is completely absent from peripheral T cells and most T cell lines based on sensitive RT-PCR assays. In contrast, Spi-B is expressed exclusively in lymphoid cells and can be detected in early fetal thymus and spleen. In situ hybridizations of adult murine tissues demonstrate Spi-B mRNA in the medulla of the thymus, the white pulp of the spleen, and the germinal centers of lymph nodes. Spi-B expression is very abundant in B cells and both Spi-B mRNA and protein are detected in some T cells. In situ hybridization and Northern blot analysis suggest that Spi-B gene expression increases during B cell maturation and decreases during T cell maturation. Gel-retardation experiments show that Spi-B can bind to all putative PU.1 binding sites, but do not reveal any preferred Spi-B binding site. Finally, both PU.1 and Spi-B function as transcriptional activators of the immunoglobulin light-chain enhancer E lambda 2.4 when coexpressed with Pip (PU.1-interaction partner) in NIH-3T3 cells. Taken together, these data suggest that differences in patterns of expression between Spi-B and PU.1 distinguish the function of each protein during development of the immune system.
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Linfocitos B/fisiología , Proteínas de Unión al ADN/metabolismo , Linfocitos T/fisiología , Transactivadores , Factores de Transcripción/metabolismo , Secuencia de Bases , Western Blotting , Línea Celular , Cartilla de ADN/química , Proteínas de Unión al ADN/genética , Elementos de Facilitación Genéticos , Regulación del Desarrollo de la Expresión Génica , Hematopoyesis , Humanos , Cadenas kappa de Inmunoglobulina/genética , Hibridación in Situ , Datos de Secuencia Molecular , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/genética , Secuencias Reguladoras de Ácidos Nucleicos , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Factores de Transcripción/genéticaRESUMEN
Calponin-h1 is a 34-kDa myofibrillar thin filament, actin-binding protein that is expressed exclusively in smooth muscle cells (SMCs) in adult animals. To examine the molecular mechanisms that regulate SMC-specific gene expression, we have examined the temporal, spatial, and cell cycle-regulated patterns of expression of calponin-h1 gene expression and isolated and structurally characterized the murine calponin-h1 gene. Calponin-h1 mRNA is expressed exclusively in SMC-containing tissues in adult animals. During murine embryonic development, calponin-h1 gene expression is (i) detectable in E9.5 embryos in the dorsal aorta, cardiac outflow tract, and tubular heart, (ii) sequentially up-regulated in SMC-containing tissues, and (iii) down-regulated to non-detectable levels in the heart during late fetal development. In addition, the gene is expressed in resting rat aortic SMCs, but its expression is rapidly down-regulated when growth-arrested cells reenter phase G1 of the cell cycle and proliferate. Calponin-h1 is encoded by a 10.7-kilobase single copy gene composed of seven exons, which is part of a multigene family. Transient transfection analyses demonstrated that 1.5 kilobases of calponin-h1 5'-flanking sequence is sufficient to program high level transcription of a luciferase reporter gene in cultured primary rat aortic SMCs and the smooth muscle cell line, A7r5. Taken together, these data suggest that the calponin-h1 gene will serve as an excellent model system with which to examine the molecular mechanisms that regulate SMC lineage specification, differentiation, and phenotypic modulation.
Asunto(s)
Proteínas de Unión al Calcio/genética , Regulación del Desarrollo de la Expresión Génica , Músculo Liso Vascular/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Evolución Biológica , Proteínas de Unión al Calcio/metabolismo , Línea Celular , Clonación Molecular , ADN Complementario , Humanos , Ratones , Proteínas de Microfilamentos , Datos de Secuencia Molecular , Familia de Multigenes , Músculo Liso Vascular/citología , Músculo Liso Vascular/fisiología , Fenotipo , Regiones Promotoras Genéticas , Ratas , Homología de Secuencia de Aminoácido , Transcripción Genética , CalponinasRESUMEN
SM22 alpha is expressed exclusively in smooth muscle-containing tissues of adult animals and is one of the earliest markers of differentiated smooth muscle cells (SMCs). To examine the molecular mechanisms that regulate SMC-specific gene expression, we have isolated and structurally characterized the murine SM22 alpha gene. SM22 alpha is a 6.2-kilobase single copy gene composed of five exons. SM22 alpha mRNA is expressed at high levels in the aorta, uterus, lung, and intestine, and in primary cultures of rat aortic SMCs, and the SMC line, A7r5. In contrast to genes encoding SMC contractile proteins, SM22 alpha gene expression is not decreased in proliferating SMCs. Transient transfection experiments demonstrated that 441 base pairs of SM22 alpha 5'-flanking sequence was necessary and sufficient to program high level transcription of a luciferase reporter gene in both primary rat aortic SMCs and A7r5 cells. DNA sequence analyses revealed that the 441-base pair promoter contains two CArG/SRF boxes, a CACC box, and one potential MEF-2 binding site, cis-acting elements which are each important regulators of striated muscle transcription. Taken together, these studies have identified the murine SM22 alpha promoter as an excellent model system for studies of developmentally regulated, lineage-specific gene expression in SMCs.
Asunto(s)
Proteínas de Microfilamentos , Proteínas Musculares/genética , Músculo Liso Vascular/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Ciclo Celular/genética , Células Cultivadas , Clonación Molecular , ADN Complementario , Ratones , Datos de Secuencia Molecular , Músculo Liso Vascular/citología , Regiones Promotoras Genéticas , RatasRESUMEN
The unique contractile phenotype of cardiac myocytes is determined by the expression of a set of cardiac-specific genes. By analogy to other mammalian developmental systems, it is likely that the coordinate expression of cardiac genes is controlled by lineage-specific transcription factors that interact with promoter and enhancer elements in the transcriptional regulatory regions of these genes. Here, we demonstrate that the slow/cardiac-specific troponin C (cTnC) enhancer contains a specific binding site for the lineage-restricted, zinc finger transcription factor, GATA-4 and that GATA-4 mRNA and protein is expressed in cardiac myocytes. In addition, GATA-4 binding sites were identified in several previously characterized cardiac-specific transcriptional regulatory elements. The cTnC GATA-4 binding site is required for transcriptional enhancer activity in primary cardiac myocytes. Moreover, the cTnC enhancer can be transactivated by over-expression of GATA-4 in non-cardiac muscle cells such as NIH 3T3 cells. Taken together, these results are consistent with a model in which GATA-4 functions to direct tissue-specific gene expression during mammalian cardiac development.
Asunto(s)
Proteínas de Unión al ADN/genética , Contracción Miocárdica/genética , Factores de Transcripción/genética , Troponina/genética , Células 3T3 , Animales , Secuencia de Bases , Proteínas de Unión al ADN/metabolismo , Factor de Transcripción GATA4 , Técnicas de Transferencia de Gen , Ratones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Factores de Transcripción/metabolismo , Activación Transcripcional , Troponina/metabolismo , Troponina CRESUMEN
The unique contractile phenotype of cardiac myocytes is determined by the expression of a set of cardiac muscle-specific genes. By analogy to other mammalian developmental systems, it is likely that the coordinate expression of cardiac genes is controlled by lineage-specific transcription factors that interact with promoter and enhancer elements in the transcriptional regulatory regions of these genes. Although previous reports have identified several cardiac muscle-specific transcriptional elements, relatively little is known about the lineage-specific transcription factors that regulate these elements. In this report, we demonstrate that the slow/cardiac muscle-specific troponin C (cTnC) enhancer contains a specific binding site for the lineage-restricted zinc finger transcription factor GATA-4. This GATA-4-binding site is required for enhancer activity in primary cardiac myocytes. Moreover, the cTnC enhancer can be transactivated by overexpression of GATA-4 in non-cardiac muscle cells such as NIH 3T3 cells. In situ hybridization studies demonstrate that GATA-4 and cTnC have overlapping patterns of expression in the hearts of postimplantation mouse embryos and that GATA-4 gene expression precedes cTnC expression. Indirect immunofluorescence reveals GATA-4 expression in cultured cardiac myocytes from neonatal rats. Taken together, these results are consistent with a model in which GATA-4 functions to direct tissue-specific gene expression during mammalian cardiac development.