RESUMEN
The distribution of ribosomal transcripts in the plant nucleolus has been studied by non-isotopic in situ hybridization in ultrathin Lowicryl K4M sections and by high-resolution autoradiography after labelling with tritiated uridine. In parallel, cytochemical techniques were applied to localize RNA on different plant nucleolar components of Allium cepa L. root meristematic cells and Capsicum annuum L. pollen grains. For RNA/RNA in situ hybridization, several biotinylated single-stranded ribosomal RNA probes were used for mapping different fragments of the 18 S and the 25 S rRNA gene transcribed regions. Ribosomal RNAs (from pre-rRNAs to mature 18 and 25 S RNAs) were found in the nucleolus, in the dense fibrillar (DFC) and granular components (GC). Hybridization signal was found at the periphery of some fibrillar centres (FCs) with probes recognizing both 18 and 25 S rRNA sequences. A quantitative study was performed to analyze the significance of this labelling. Incorporation of tritiated uridine into roots was carried out and, later, after a long time-exposure, autoradiography revealed the presence of newly synthesized RNA mainly in the DFC and at the periphery of the FCs. The presence of RNA in these areas was also confirmed by the cytochemical techniques used in this study. Taken together, these data favour the hypothesis that transcription can begin at the periphery of the FCs, although we cannot exclude the possibility that the DFC plays a role in this process.
Asunto(s)
Nucléolo Celular/ultraestructura , ARN Mensajero/ultraestructura , ARN Ribosómico/ultraestructura , Verduras/ultraestructura , Allium/ultraestructura , Arabidopsis/genética , Autorradiografía , Capsicum/ultraestructura , ADN Ribosómico/genética , Histocitoquímica/métodos , Hibridación in Situ , Plantas Medicinales , Sondas ARN , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , ARN Ribosómico/genética , ARN Ribosómico/aislamiento & purificación , ARN Ribosómico 18S/genética , ARN Ribosómico 18S/aislamiento & purificación , ARN Ribosómico 18S/ultraestructura , Transcripción Genética , Verduras/genéticaRESUMEN
It was previously shown that a 1.5 kb fragment located in the non-transcribed spacer (NTS) is the earliest replicating region of pea (Pisum sativum) rDNA in synchronized root cells. In the present report the structure of this region was characterized. It contains a cluster of four 11 bp near matches to the Saccharomyces cerevisiae ARS consensus sequence (ACS). These near matches are embedded in an A+T rich domain located upstream from the transcription initiation site. We identified and mapped an intrinsic DNA bending locus 5' to the cluster of near matches. Several eukaryotic origins including the ARS from the budding yeast show very similar structural features. This observation strengthens the notion that pea rDNA replication initiates at or near this region. Replication of the entire pea rDNA repeat was analysed by two-dimensional (2D) agarose gel electrophoresis. The results obtained indicate that only a small fraction of the potential origins is used in each replication round. Forks moving in the direction opposite to rRNA transcription are stalled at a polar replication fork barrier (RFB), which mapped near the 3' end of the transcription unit. Consequently, most of pea rDNA appears to replicate in a unidirectional manner. These results show that the strategy used to replicate pea and yeast rRNA genes is very similar, suggesting that it has been conserved and might be common to most eukaryotes.