RESUMEN
Fixed cells with different nucleic acid contents and scatter properties (low nucleic acid [LNA], high nucleic acid 1 [HNA1], and HNA2) were sorted by flow cytometry (FCM). For each sort, 10,000 cells were efficiently captured on poly-l-lysine-coated microplates, resulting in efficient and reproducible PCR amplification.
Asunto(s)
Bacterias/aislamiento & purificación , Separación Celular/métodos , Citometría de Flujo/métodos , Bacterias/genética , Datos de Secuencia Molecular , Técnicas de Amplificación de Ácido Nucleico/métodos , Ácidos Nucleicos/metabolismo , Reacción en Cadena de la Polimerasa/métodos , ARN Ribosómico 16S/genética , Agua de Mar/microbiologíaRESUMEN
Dinoflagellates are unique among eukaryotes in their lack of histones and nucleosomes, and permanently condensed chromosomes. These unusual features raise questions as how chromatin condensation and gene expression are achieved. In this study, we investigated nuclear proteins potentially implicated in the regulation of the transcription. Dinap1 is a dinoflagellate nuclear protein that has a WW domain and is synthesized mainly in G1 and S phases of the cell cycle. In this study, we found that Dip1, a proline-rich potential ligand of Dinap1, and DapC, a Dip1 potential ligand, were both present in the nucleus of Crypthecodinium cohnii during the G1 phase. Dip1 contained a PPXY motif, and its domain organization was similar to that of the splicing factor FBP21 in that it possessed one zinc finger and two WW domains. Although DapC has no known homolog, 22 repeats of a PPXPXGX heptapeptide were identified at the N-terminus, and this structure is similar to that of the C-terminal part of the mouse splicing factor SAP62. Dinap1 was co-precipitated with Dip1 and DapC in vitro and in vivo, but despite their nuclear location, these three proteins did not bind directly to DNA. Dinap1 activated up to 40% of the basal transcription activity of C. cohnii in an in vitro assay, whereas DapC inhibited it by 40% and Dip1 had no effect. These dinoflagellate proteins appear to be the subunits of a nuclear complex that may be involved in regulating transcription.
Asunto(s)
Dinoflagelados/metabolismo , Proteínas de Drosophila , Regulación de la Expresión Génica , Secuencias Hélice-Asa-Hélice , Proteínas Nucleares/metabolismo , Péptidos/metabolismo , Proteínas Protozoarias/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Núcleo Celular/metabolismo , ADN/metabolismo , Dinoflagelados/genética , Humanos , Ligandos , Ratones , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Fosforilación , Prolina , Dominios Proteicos Ricos en Prolina , Estructura Terciaria de Proteína , Proteínas Protozoarias/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
The morphology and behaviour of the chromosomes of dinoflagellates during the cell cycle appear to be unique among eukaryotes. We used synchronized and aphidicolin-blocked cultures of the dinoflagellate Crypthecodinium cohnii to describe the successive morphological changes that chromosomes undergo during the cell cycle. The chromosomes in early G(1) phase appeared to be loosely condensed with numerous structures protruding toward the nucleoplasm. They condensed in late G(1), before unwinding in S phase. The chromosomes in cells in G(2) phase were tightly condensed and had a double number of arches, as visualised by electron microscopy. During prophase, chromosomes elongated and split longitudinally, into characteristic V or Y shapes. We also used confocal microscopy to show a metaphase-like alignment of the chromosomes, which has never been described in dinoflagellates. The metaphase-like nucleus appeared flattened and enlarged, and continued to do so into anaphase. Chromosome segregation occurred via binding to the nuclear envelope surrounding the cytoplasmic channels and microtubule bundles. Our findings are summarized in a model of chromosome behaviour during the cell cycle.