RESUMO
Cyclins are regulatory proteins that interact with cyclin-dependent kinases (CDKs) to control progression through the cell cycle. In Arabidopsis thaliana, 34 cyclin genes have been described, grouped into five different types (A, B, D, H, and T). A novel class of seven cyclins was isolated and characterized in Arabidopsis, designated P-type cyclins (CYCPs). They all share a conserved central region of 100 amino acids ("cyclin box") displaying homology to the corresponding region of the PHO80 cyclin from Saccharomyces cerevisiae and the related G1 cyclins from Trypanosoma cruzi and T. brucei. The CYCP4;2 gene was able to partially re-establish the phosphate-dependent expression of the PHO5 gene in a pho80 mutant strain of yeast. The CYCPs interact preferentially with CDKA;1 in vivo and in vitro as shown by yeast two-hybrid analysis and co-immunoprecipitation experiments. P-type cyclins were mostly expressed in proliferating cells, albeit also in differentiating and mature tissues. The possible role of CYCPs in linking cell division, cell differentiation, and the nutritional status of the cell is discussed.
Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Quinases Ciclina-Dependentes/química , Ciclinas/química , Ciclinas/genética , Regulação da Expressão Gênica de Plantas , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas de Saccharomyces cerevisiae/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Arabidopsis , Proteínas de Arabidopsis/metabolismo , Diferenciação Celular , Núcleo Celular/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Citoplasma/metabolismo , Teste de Complementação Genética , Hibridização In Situ , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Fosfatos/química , Filogenia , Plasmídeos/metabolismo , Testes de Precipitina , Ligação Proteica , Biossíntese de Proteínas , Estrutura Terciária de Proteína , RNA/química , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas de Saccharomyces cerevisiae/química , Homologia de Sequência de Aminoácidos , Transcrição Gênica , Técnicas do Sistema de Duplo-HíbridoRESUMO
Sedentary edoparasitic nematodes induce specialised feeding cells in plant roots. Giant cells induced by root knot nematodes and syncytia generated by cyst nematodes in plant roots are large multinucleated cells containing a dense cytoplasm. To examine the plant cytoskeleton during feeding cell development, transcriptional activity of actin and tubulin genes and organization of the actin filaments and of the microtubules were analyzed in situ. Immunolocalizations of actins and tubulins and in vivo observation of green fluorescent protein decorated actin filaments and microtubules in nematode infected root cells revealed that major rearrangements of the cytoskeleton occur during the formation of nematode induced feeding cells.
Assuntos
Actinas/genética , Arabidopsis/genética , Citoesqueleto/ultraestrutura , Nematoides/anatomia & histologia , Nematoides/fisiologia , Tubulina (Proteína)/genética , Ração Animal , Animais , Arabidopsis/parasitologia , Proteínas de Fluorescência Verde , Imuno-Histoquímica , Proteínas Luminescentes/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/parasitologia , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/metabolismo , Sementes/parasitologiaRESUMO
In situ hybridization detection of mRNA is an essential tool for understanding regulation of gene expression in cells and tissues of different organisms. Over the years, numerous in situ protocols have been developed ranging from whole-mount techniques that allow fast transcript localization in intact organs to high-resolution methods based on the electron microscopic detection of mRNAs at the subcellular level. Here, we present a detailed protocol for the detection of mRNAs in plant tissues using radiolabeled single-stranded RNA probes. Hybridizations are carried out on tissue sections of paraffin- and plastic-embedded plant tissues. Although this in situ protocol is appropriate for plant tissues in general, it has been optimized for Arabidopsis thaliana. Variations on the procedure, required to obtain optimal results with different Arabidopsis tissues, are described.