Faro-Trindade Inês, Cook Peter R
Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, United Kingdom.
Mol Biol Cell. 2006 Jul;17(7):2910-20. doi: 10.1091/mbc.e05-11-1024. Epub 2006 Apr 19.
Although we have detailed information on the alterations occurring in steady-state levels of all cellular mRNAs during differentiation, we still know little about more global changes. Therefore, we investigated the numbers of molecules of RNA polymerase II that are active--and the way those molecules are organized--as two mouse cells (aneuploid F9 teratocarcinoma, and euploid and totipotent embryonic stem cells) differentiate into parietal endoderm. Quantitative immunoblotting shows the number of active molecules roughly halves. Transcription sites (detected by light and electron microscopy after allowing engaged polymerases to extend nascent transcripts in bromouridine-triphosphate) are uniformly distributed throughout the nucleoplasm. The numbers of such sites fall during differentiation as nuclei become smaller, but site density and diameter remain roughly constant. Similar site densities and diameters are found in salamander (amphibian) cells with 11-fold larger genomes, and in aneuploid HeLa cells. We conclude that active polymerases and their nascent transcripts are concentrated in a limited number of discrete nucleoplasmic sites or factories, and we speculate that the organization of transcription is conserved during both differentiation and evolution to a high C value.
尽管我们已经掌握了细胞分化过程中所有细胞mRNA稳态水平变化的详细信息，但对于更全局性的变化我们仍然知之甚少。因此，我们研究了两种小鼠细胞（非整倍体F9畸胎瘤细胞以及整倍体全能胚胎干细胞）分化为滋养层内胚层时，活跃的RNA聚合酶II分子数量以及这些分子的组织方式。定量免疫印迹显示活跃分子数量大致减半。转录位点（在溴尿苷三磷酸中使结合的聚合酶延伸新生转录本后，通过光学显微镜和电子显微镜检测）均匀分布于整个核质中。随着细胞核变小，分化过程中转录位点的数量减少，但位点密度和直径大致保持不变。在基因组大11倍的蝾螈（两栖动物）细胞以及非整倍体HeLa细胞中也发现了类似的位点密度和直径。我们得出结论，活跃的聚合酶及其新生转录本集中在有限数量的离散核质位点或工厂中，并且我们推测转录的组织方式在分化和进化至高C值的过程中是保守的。
