多头绒泡菌间期细胞核中RNA的转录状况

利用BrUTP免疫标记技术,研究了多头绒泡菌（Physarum polycephalum Sclhw.）间期细胞核中RNA的转录状况。结果表明：在整修间期核仁中的rRNA都在活跃转录;核质中hnRNA的转录呈逐渐上升趋势,早S期转录水平很低,晚S期转录活性升高1倍,G2期转录达到最高水平;整修间期核质中RNA的转录水平增加了5－6倍。     

Regulation of c-myc through intranuclear localization of its RNA subspecies

We used fluorescence in situ hybridization (FISH) to detect c-myc RNA subspecies in human COLO 320DM tumor cells. Although the FISH procedure removed the majority of RNAs from the nucleolus, c-myc RNA continued to be detected in both the nucleoplasm and nucleolus. This finding suggests stable association between c-myc RNA and the nucleolus. Nucleolar accumulation of c-myc RNA appeared to be temporally regulated by cell-cycle progression. Hybridization with exon- and strand-specific RNA probes indicated that the non-protein coding exon 1 plays a novel role in determining the subnuclear localization of c-myc RNA. Antisense RNA targeting exon 2 localized only with nucleoplasmic foci, where it might interact with the sense strand. Thus, c-myc gene expression may be regulated by intranuclear localization of its RNA.     

Mammalian nuclei contain foci which are highly enriched in components of the pre-mRNA splicing machinery.

The organization of the major snRNP particles in mammalian cell nuclei has been analysed by in situ labelling using snRNA-specific antisense probes made of 2'-OMe RNA. U3 snRNA is exclusively detected in the nucleolus while all the spliceosomal snRNAs are found in the nucleoplasm outside of nucleoli. Surprisingly, U2, U4, U5 and U6 snRNAs are predominantly observed in discrete nucleoplasmic foci. U1 snRNA is also present in foci but in addition is detected widely distributed throughout the nucleoplasm. An anti-peptide antibody specific for the non-snRNP splicing factor U2AF reveals it to have a similar distribution to U1 snRNA. Co-localization studies using confocal fluorescence microscopy prove that U2AF is present in the snRNA-containing foci. Antibody staining also shows the foci to contain snRNP-specific proteins and m3G-cap structures. The presence of major components of the nuclear splicing apparatus in foci suggests that these structures may play a role in pre-mRNA processing.     

栽培玉米亚种5 S rDNA NTS序列分析及FISH定位方法比较

选取我国7个栽培玉米亚种材料,进行5 S rDNA的非转录间隔区（nontranscribed intergenic spacer,NTS）的序列分析,比较7个亚种材料NTS序列差异并进行聚类分析,探讨其亲缘关系。研究结果表明：7个材料的NTS区GC平均含量为45．67％,核苷酸位点变异位点个数1～15,转换／颠换率为0．83～2．0,特用玉米材料均存在不同程度的缺失;7个材料主要聚为两大类,第一类群中包括甜质、马齿、硬粒、爆裂和蜡质5个亚种材料,第二类群中包括粉质和甜粉2个亚种材料。同时利用荧光原位杂交技术（fluorescence in situ hybridization,FISH）对5 S rDNA进行定位,探针标记分别采用荧光素标记和生物素标记。结果表明：生物素标记检测系统灵敏度高、杂交信号强,更适合于5 S rDNA重复序列的定位检测。     

Membrane labelling by fluorescent probes: incorporation of TMA-DPH in erythrocyte membranes.

A study of the labelling of isolated resealed erythrocyte membranes by TMA-DPH has been carried out. A quantitative study shows that saturation appears to take place when increasing the relative quantity of probe bulk concentration to membrane concentration; this is readily interpreted by a simple incorporation model with a limited number of sites in the membrane. A qualitative study shows that an increase in the labelling leads to an evolution of the probe fluorescence properties; the existence of different types of sites is involved in the interpretation but the system is too complex to allow it to be represented by a simple model. As a consequence of this study, care has to be taken in labelling biological material so as to avoid excessive probe incorporation.     

Dynamics and three-dimensional localization of ribosomal RNA within the nucleolus

Although rRNA synthesis, maturation, and assembly into preribosomal particles occur within the nucleolus, the route taken by pre-rRNAs from their synthetic sites toward the cytoplasm remains largely unexplored. Here, we employed a nondestructive method for the incorporation of BrUTP into the RNA of living cells. By using pulse-chase experiments, three-dimensional image reconstructions of confocal optical sections, and electron microscopy analysis of ultrathin sections, we were able to describe topological and spatial dynamics of rRNAs within the nucleolus. We identified the precise location and the volumic organization of four typical subdomains, in which rRNAs are successively moving towards the nucleolar periphery during their synthesis and processing steps. The incorporation of BrUTP takes place simultaneously within several tiny spheres, centered on the fibrillar centers. Then, the structures containing the newly synthesized RNAs enlarge and appear as compact ringlets disposed around the fibrillar centers. Later, they form hollow spheres surrounding the latter components and begin to fuse together. Finally, these structures widen and form large rings reaching the limits of the nucleoli. These results clearly show that the transport of pre-rRNAs within the nucleolus does not occur randomly, but appears as a radial flow starting from the fibrillar centers that form concentric rings, which finally fuse together as they progress toward the nucleolar periphery.