RESUMO
Rabbit polyclonal antibodies were raised against the C-terminal fragment (amino acid residues 318-451) of human gamma-tubulin. These antibodies were used to stain cultured cells of various tissues (epithelium, nervous tissue, fibroblasts) from different animals (human, monkey, pig, rat, kangaroo rat, mouse, hamster, chicken, triton). The antibodies specifically stained centrioles in the interphase and mitotic cells of mammals, but not birds (chicken) or amphibians (newt). In the interphase cells, centrioles were stained as a pair of dots (or as a double dot) in 96-97% of the cells. The distances between the maternal and filial centrioles varied in different cultures. Procentrioles were stained in certain cells, but with less intensity than mature centrioles. In mitotic cells, the antibodies revealed two spots corresponding to two mitotic poles. The spots in mitosis were significantly larger than the interphase dots, but the staining was more faint. In spontaneous tripolar mitoses, only two poles were stained. Thus, it was shown that, on the one hand, gamma-tubulin is associated with centrioles irrespective of whether or not they serve as the microtubule organizing centres and, on the other hand, gamma-tubulin might not be an essential component of the microtubule organizing centres.
Assuntos
Anticorpos , Centríolos/metabolismo , Tubulina (Proteína)/imunologia , Tubulina (Proteína)/metabolismo , Animais , Células Cultivadas , Galinhas , Chlorocebus aethiops , Cricetinae , Cricetulus , Células Epiteliais , Epitélio/metabolismo , Epitélio/ultraestrutura , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Células HeLa , Humanos , Immunoblotting , Interfase/fisiologia , Macropodidae , Camundongos , Microtúbulos/metabolismo , Mitose/fisiologia , Tecido Nervoso/citologia , Tecido Nervoso/metabolismo , Tecido Nervoso/ultraestrutura , Ratos , Salamandridae , Coloração e Rotulagem/métodos , Suínos , Células VeroAssuntos
Vírus de Plantas/genética , Proteínas do Envelope Viral/genética , Sequência de Aminoácidos , Sequência de Bases , Western Blotting , Cromatografia em Agarose , Clonagem Molecular , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Técnicas Imunoenzimáticas , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Proteínas do Envelope Viral/isolamento & purificação , Proteínas do Envelope Viral/metabolismoRESUMO
The 5'-terminal fragment (containing 1-565th codons) of Bacillus thuringiensis var. tenebrionis gene for the Coleoptera-specific delta-endotoxin CryIIIA was cloned. This sequence was extended with either only a homologous fragment of CryIA(a) or that one together with in-frame NPTII or GUS coding sequences. The obtained gene derivatives were expressed in E. coli. The analysis of hybrid polypeptides confirmed the enzymatic activities of bifunctional proteins and showed toxic properties of "insectotoxin-NPTII" against Colorado potato beetle (Leptinotarsa decemlineata).
