The Studies On Dynamic Changes Between Disruption And Restoration Of Nucleolar Ultrastructure And Nucleolar RDNA Distribution And Transcription During The Cell Cycle In HeLa Cells

The nucleolus is the most significant structure in the interphase nuclei,and the place of gene transcription of product ribosomal RNA and further processing and maturity of the transcription product.The nucleolus is mainly composed of fibrillar center(FC),dense fibrillar component(DFC)and granular component(GC).At the same time,the nucleolus regularly breaks up,disappears and reconstructs in the cell cycle.Due to the characteristic of highly dynamic change,the research process of the ultrastructure and functions of nucleolus is slow.It is also one of the important reasons of many debates.The main focus of the argument is the specific area for the distribution of ribosomal gene(r DNA)in the nucleolus,transcription locus and the splice site where r RNA precursor is formed after transcription.In the past most researchers only conducted the static research on the cell Nucleolus in a certain period in the cell cycle,but ignored the structure with dynamic change.Even in the relatively stable interphase in cell division,its ultrastructure also changes with the process of G1,S and G2 phase,so r DNA distribution,transcription and splice site also should dynamically change.This paper used the thymidine double blocking method for the synchronized culture of HeLa cells.PCNA immune antibody marking,the specific staining method of acridine orange and conventional electron microscope ultra-thin slicing technology were combined with the NAMA-Ur specificity method,Bernhard staining method and other methods to study the ultrastructural changes of HeLa cell nucleolus in different periods of the cell cycle,the distribution of r DNA in nucleolus and transcription locus.The main results are as follows:1.The number,size and shape of FC and DFC in the ultrastructure of HeLa nucleolus have a series of complicated dynamic variation function in the cell cycle to satisfy the needs of functions of the nucleolus.2.The specific staining of r DNA was completed with the NAMA-Ur method.According to the results,r DNA's dense chromatin mass and loose cloud-shaped DNA components have different distribution positions and shapes in different periods in the cell cycle.3.The nucleolar RNA was preferentially stained by Bernhard staining.The results showed that the marking of r RNA transcription site of HeLa cell nucleolus appeared at the junction of FC and DFC and in DFC.In different periods in the cell interphase,the results were also different.4.DNA and RNA of HeLa cell nucleolus were specially stained with acridine orange.The results showed that in the G2 phase the fluorescent signal of RNA orange was the strongest,which indicated that in the G2 phase the RNA transcription activity was the strongest.5.The B23 protein immune antibody marker was used for observation under the light microscope.The results suggested that in G1 phase,S phase and G2 phase B23 protein always existed,particularly in the G2 phase B23 protein marker had the strongest fluorescent signal,and in the division phase in the cell nucleolus the obvious B23 protein fluorescence signal could be hardly observed,which indicated that in the HeLa cell nucleolus B23 protein in the active state and storage state existed.In conclusion,the number and shape of FC and DFC in the structure of HeLa cell nucleolus also have highly dynamic changes in the interphase of cell division.Especially in the middle S phase FC and DFC of the nucleolus had the morphological changes of reorganization and restructuring.This is our new finding.In the interphase of cell division,r DNA of the nucleolus was distributed in FC and DFC area in the form of dense chromatin mass and loose cloud-shaped DNA,and had dynamic changes.It shows that RNA transcription of nucleolus and activity state and site of precursor splicing constantly change in the G1,S and G2 phase of interphase of cell division.The observation results of this experiment provide and improve the experimental basis and data basis,in order to further clarify the relationship between the ultrastructure,RNA transcription and splicing of the eukaryotic cell nucleolus in the process of cell cycle.