RESUMO
CA150 represses RNA polymerase II (RNAPII) transcription by inhibiting the elongation of transcripts. The FF repeat domains of CA150 bind directly to the phosphorylated carboxyl-terminal domain of the largest subunit of RNAPII. We determined that this interaction is required for efficient CA150-mediated repression of transcription from the alpha(4)-integrin promoter. Additional functional determinants, namely, the WW1 and WW2 domains of CA150, were also required for efficient repression. A protein that interacted directly with CA150 WW1 and WW2 was identified as the splicing-transcription factor SF1. Previous studies have demonstrated a role for SF1 in transcription repression, and we found that binding of the CA150 WW1 and WW2 domains to SF1 correlated exactly with the functional contribution of these domains for repression. The binding specificity of the CA150 WW domains was found to be unique in comparison to known classes of WW domains. Furthermore, the CA150 binding site, within the carboxyl-terminal half of SF1, contains a novel type of proline-rich motif that may be recognized by the CA150 WW1 and WW2 domains. These results support a model for the recruitment of CA150 to repress transcription elongation. In this model, CA150 binds to the phosphorylated CTD of elongating RNAPII and SF1 targets the nascent transcript.
Assuntos
Proteínas de Ligação a DNA , RNA Polimerase II/metabolismo , Precursores de RNA/metabolismo , Splicing de RNA , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/metabolismo , Transativadores/metabolismo , Fatores de Transcrição , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular Transformada , Regulação da Expressão Gênica , Células HeLa , Humanos , Dados de Sequência Molecular , Fosforilação , Prolina/metabolismo , Fatores de Processamento de RNA , Proteínas de Ligação a RNA/genética , Proteínas Repressoras/genética , Relação Estrutura-Atividade , Transativadores/genética , Fatores de Elongação da TranscriçãoRESUMO
Human immunodeficiency virus-1 Tat protein and human Cyclin T1 mediate transcriptional activation by enhancing the elongation efficiency of RNA polymerase II. Activation of transcription of the related equine infectious anemia virus (EIAV) requires a similar protein known as eTat, which does not function in human cells. Expression of equine Cyclin T1 in human cells rescues eTat function, suggesting a general mechanism of transcription activation among lentiviruses. Here we present the cloning of Cyclin T1 from canine D17 osteosarcoma cells, which support EIAV transactivation, and show that canine Cyclin T1 confers eTat transactivation to human cells. A two-amino-acid change, from 79-proline-glycine-80 to 79-histidine-arginine-80, confers on the human Cyclin T1 the ability to cooperate with eTat in transcriptional activation. These findings suggested that the regions of Cyclin T1 that interact with lentiviral Tat proteins and TAR RNA elements form an extended domain, which very likely has a conserved fold.
Assuntos
Ciclinas/metabolismo , Produtos do Gene tat/genética , Produtos do Gene tat/metabolismo , Vírus da Anemia Infecciosa Equina/genética , Ativação Transcricional , Sequência de Aminoácidos , Animais , Clonagem Molecular , Ciclina T , Ciclinas/química , Ciclinas/genética , Cães , Humanos , Vírus da Anemia Infecciosa Equina/metabolismo , Camundongos , Dados de Sequência Molecular , RNA Viral/genética , Elementos de Resposta/genética , Sequências Repetidas Terminais/genética , Células Tumorais CultivadasRESUMO
Immunohistochemical evaluation of equine tissue necessitates the use of antibodies reactive with cells from a heterogeneous population. Lymphoid tissues from 12 horses were fixed in Bouin's fluid, ethanol or formalin and examined for immunohistochemical reactivity with anti-equine and anti-human monoclonal antibodies (MAbs) specific for MHC Class II antigens, T and B lymphocytes, and macrophages. Only a few of the anti-equine MAbs tested were reactive with fixed, paraffin wax-embedded tissue. Anti-human MAbs expanded the desired range of reactivity and increased the consistency in different animals. The anti-equine MAbs conferred species specificity and anti-human MAbs provided an increased spectrum of reactivity.
Assuntos
Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos/imunologia , Cavalos/imunologia , Imuno-Histoquímica , Ácido Acético/imunologia , Animais , Linfócitos B/imunologia , Etanol/imunologia , Formaldeído/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Linfonodos/imunologia , Tonsila Palatina/imunologia , Picratos/imunologia , Linfócitos T/imunologia , Fixação de TecidosRESUMO
The atomic force microscope (AFM) can be used to image the surface of both conductors and nonconductors even if they are covered with water or aqueous solutions. An AFM was used that combines microfabricated cantilevers with a previously described optical lever system to monitor deflection. Images of mica demonstrate that atomic resolution is possible on rigid materials, thus opening the possibility of atomic-scale corrosion experiments on nonconductors. Images of polyalanine, an amino acid polymer, show the potential of the AFM for revealing the structure of molecules important in biology and medicine. Finally, a series of ten images of the polymerization of fibrin, the basic component of blood clots, illustrate the potential of the AFM for revealing subtle details of biological processes as they occur in real time.
Assuntos
Cristalografia , Microscopia/instrumentação , Polímeros , Água , Fibrina , Humanos , Técnicas In Vitro , Peptídeos , Trombina , Gravação em VídeoRESUMO
Atomic force microscope images of polymerized monolayers of n-(2-aminoethyl)-10,12-tricosadiynamide revealed parallel rows of molecules with a side-by-side spacing of approximately equal to 0.5 nanometer. Forces used for imaging (10(-8) newton) had no observable effect on the polymer strands. These results demonstrate that atomic force microscope images can be obtained for an organic system.