RESUMO
The specificity and structural simplicity of the bacteriophage T3, T7, and SP6 RNA polymerases make these enzymes particularly well suited for studies of polymerase-promoter interactions. To understand the initial recognition process between the enzyme and its promoters, DNA fragments that carry phage promoters were chemically modified by three different methods: base methylation, phosphate ethylation, and base removal. The positions at which these modifications prevented or enhanced binding by the RNA polymerases were then determined. The results indicate that specific contacts within the major groove of the promoter between positions-5 and -12 are important for phage polymerase binding. Removal of individual bases from either strand of the initiation region (-5 to +3) resulted in enhanced binding of the polymerase, suggesting that disruption of the helix in this region may play a role in stabilization of the polymerase-promoter complexes.
Assuntos
Colífagos/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Regiões Promotoras Genéticas , Fagos T/genética , Sequência de Bases , Colífagos/enzimologia , Escherichia coli/enzimologia , Metilação , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Ligação Proteica , Homologia de Sequência do Ácido Nucleico , Fagos T/enzimologiaRESUMO
We describe a method for specifically labelling T7 RNA polymerase at (or near) the active site. Enzyme molecules that have been modified by covalent attachment of a benzaldehyde nucleotide derivative in the presence of template DNA are subsequently incubated with radioactively labelled nucleoside triphosphates. Labelling of the enzyme occurs as a result of the formation of the first phosphodiester bond. The labelling is template-directed and the expected specificity of initiation at individual T7 promoters is observed. The label has been localized to an 80 kd tryptic fragment that contains the carboxy-terminal portion of the enzyme.
Assuntos
DNA Polimerase Dirigida por DNA , Fagos T/enzimologia , Marcadores de Afinidade , Sítios de Ligação , Nucleotídeos de Guanina , Marcação por Isótopo/métodos , Radioisótopos de FósforoRESUMO
The hemagglutinin-neuraminidase (HN) gene sequence was determined for 16 antigenic variants of human parainfluenza virus type 3 (PIV3). The variants were selected by using monoclonal antibodies (MAbs) to the HN protein which inhibit neuraminidase, hemagglutination, or both activities. Each variant had a single-point mutation in the HN gene, coding for a single amino acid substitution in the HN protein. Operational and topographic maps of the HN protein correlated well with the relative positions of the substitutions. There was little correlation between the cross-reactivity of a MAb with the bovine PIV3 HN and the amount of amino acid homology between the human and bovine PIV3 HN proteins in the regions of the epitopes, suggesting that many of the epitopes are conformational in nature. Computer-assisted analysis of the HN protein predicted a secondary structure composed primarily of hydrophobic beta sheets interconnected by random hydrophilic coil structures. The HN epitopes were located in predicted coil regions. Epitopes recognized by MAbs which inhibit neuraminidase activity of the virus were located in a region which appears to be structurally conserved among several paramyxovirus HN proteins and which may represent the sialic cid-binding site of the HN molecule.
Assuntos
Vírus da Parainfluenza 3 Humana/imunologia , Respirovirus/imunologia , Proteínas do Envelope Viral/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais , Bovinos , Reações Cruzadas , Epitopos , Proteína HN , Hemaglutinação , Humanos , Neuraminidase/metabolismo , Testes de Neutralização , Conformação Proteica , Relação Estrutura-AtividadeAssuntos
Clonagem Molecular , Genes Virais , Genes , Hemaglutininas Virais/genética , Neuraminidase/genética , Vírus da Doença de Newcastle/genética , RNA Mensageiro/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Enzimas de Restrição do DNA , Ácido N-Acetilneuramínico , Conformação Proteica , Ácidos Siálicos/metabolismoRESUMO
The endocytosis of histones by cultured cells was examined by flow cytofluorometry. Monolayer cultures of Chinese hamster ovary cells were incubated with fluorescein-labeled histone for various periods of time and then trypsinized to remove surface-bound protein. Internalization followed first order kinetics with a half-time of 45 min, and was linear in histone concentration up to 80 microgram/ml. Since fluorescein fluorescence decreases with decreasing pH, the fluorescence of labeled histone contained in lysosomes was expected to be decreased relative to its fluorescence at neutral pH. This was demonstrated by using chloroquine to increase the lysosomal pH of intact cells. The fluorescence of labeled histones incorporated into cells increased when those cells were incubated with 50 microM chloroquine and remeasured. This provides a method for measuring the kinetics of entry of a fluorescent probe into lysosomes. Internalization into lysosomes began almost immediately upon addition of histone, but stable nonlysosomal fluorescence appeared only after a lag of 1 h. Using suspension cultures, the short term binding and internalization kinetics were also measured. In pulse-chase experiments, lysosomal fluorescence decreased with a half-time of 30 min, but nonlysosomal fluorescence decreased with a half-time of almost 12 h, probably as a result of cell division. These results demonstrate the usefulness of flow cytometry for the quantitation and characterization of endocytosis in cultured cells.
