3-DIMENSIONAL CO-LOCATION OF RNA-POLYMERASE-I AND DNA DURING INTERPHASE AND MITOSIS BY CONFOCAL MICROSCOPY

The relative three-dimensional co-location of RNA polymerase I(RPI) an d DNA was studied using confocal laser scanning microscopy during inte rphase and all the steps of mitosis in human cancerous cells. For each step of the cell cycle, immunolabeled RPI molecules and DNA specifica lly stained with chromomycin A3 were simultaneously imaged at high res olution through numerous optical sections. Then, all the data obtained were used to generate transverse sections, anaglyphs and volumic repr esentations, which are all prerequisite approaches to a representative study of the three-dimensional organization of the nucleolus and the mitotic chromosomes. Our results indicated that in the interphasic nuc lei, in which DNA is organized as a regular 3-D network. RPI was prese nt within numerous irregular spheres arranged as several twisted neckl aces. During metaphase. RPI labeling was segregated into pairs of sphe res and typical crescent-shaped structures; both were centrally locate d within the set of chromosomes. During anaphase and telophase, a typi cal central and symmetric arrangement of labeled structures was system atically seen among the decondensing chromosomes, arranged as a regula r cylinder and as a hollow half-sphere, respectively. This typical 3-D organization of structures containing RPI relative to DNA is another strong example of the nonrandom organization of the genome during inte rphase and mitosis.