Dynamic Changes Of Nucleolar Ultrastructure And The Sites Of RRNA Transcription And Processing During The Cell Cycle In Allium Sativum

The nucleolus, the ribosome factory, is the most prominent structural and functional domain in the eukaryotic nucleus. Transcription of rDNA generates 45S pre-rRNA that are subsequently cleaved and processed into 18S rRNA, 5.8S rRNA and 28S rRNA concomitant with their assembly into large and small subunits. Under the electron microscope, three basic structural compartments can be distinguished within the nucleolus: the fibrillar centers (FCs), the dense fibrillar component (DFC) and the granular component (GC). Of these components, the spherical FCs, and the DFC surrounding them, constitute a filamentous structure termed the nucleolonema. Over the years, the localization and transcription sites of rDNA and processing sites of pre-rRNA have been intensively investigated. The precise structure and functional relationship between the steps of preribosome synthesis and specific nucleolar substructures remains unclear. The incompatible results have been reported even with the same methods. But these studies were carried out on the basis that the structure of nucleolus is regarded as a static state. However, the nucleolus is a highly dynamic structure during the cell cycle. It disassembles in the late prophase and reassembles at the end of mitosis. Meanwhile the nucleolar ultrastrustructure is varying with G1, S and G2 phase during the interphase, which might be a reason for the controversy about these exact sites of transcription and processing of rRNA precursors within the nucleolus.In this report, we have used synchronized populations of Allium sativum root meristematic cells, obtained by treating root merstems with hydroxyurea (HU). Under the help of conventional transmission electron microscope, DNA-selective staing method, BrUTP pulse-labelling, immunoelectron microscopy and in situ hybridization, we studied on: the ultrastructure of nucleolus during cell cycle, the sites and arrange form of nuclolar DNA, the rRNA transcription sites, the distribution of U3 snoRNA and fibrillarin within different phases nucleolus during the cell cycle. The major results and conclusions are as fllows:1. The nucleolar ultrastructure was high dynamic change during the nucleolar cycle. The FCs and DFCs changed their size, number and features within different phase nucleoli. The cause of morphological variation is the nucleolar activity, i.e., the rate of transcription and processing of rRNA precursors.2. Using the NAMA-Ur DNA-specific staining technique showed that nucleolar rDNA is present in two forms: compacted chromatin clumps and a decondensed DNA cloud. The form of the rDNA within the nucleolus varied greatly as the cell cycle progressed. During telophase, chromosomes extended into the prenucleolar body. In early G1 phase, rDNA was only located in the fibrillar centers in the form of the condensed chromatin clump, while in mid-G1, S and G2 phases, the two forms of rDNA were distributed in FCs and DFCs. In prophase of mitosis, nucleolar rDNA, along with FC and DFC, was linked into a network structure. The area of the DNA cloud in DFC changed during different phases of the cell cycle.3. Nascent rRNA is labeled by direct incorporation of BrUTP, followed by colloidal gold particles immuno-labelling. Our results revealed that the labelling signals distributed in DFC regions around FCs within G1 phase nucleolus. In S, G2 and early prophase nucleoli, larges numbers of gold particles were located in the DFC, and the density of gold particles became higher. These results clear indicate that the part of DFC regions is the site of rRNA transcription. The statistically showed that the level of rRNA synthetic rate in DFC varies with G1, S, G2 and early prophase.4. The distribution of fibrillarin and U3 snoRNA during the cell cycle were studied by immunoelectron microscopy and in situ hybridization, the results showed that fibrillarin and U3 snoRNA localized in DFC regions, especially where it abuts the FCs within early G1 phase nucleolus. In S, G2 and early prophase nucleoli, the labelling signals were highly enriched over DFC. The GC were slightly labelled, FC were general unlabelled. These results indicate that the DFC is the major site of initial step of pre-rRNA processing. Our double-labelling experiments further proved these results, and indicated that fibrillarin and U3 snoRNA existed in two different forms within nucleolus: inactive, stored form and active form.