A 100 kDa nucleolar protein which is transitorily associated with preribosomes in the nucleoli of Chinese hamster ovary cells has been found to be specifically cleaved by a thiol protease. During an 'in vitro' incubation of nucleoli, the 100 kDa protein is processed into eight different proteins which are detected by immunoreaction with a serum raised against the 100 kDa protein. Qualitative and quantitative variations in the maturation products of the 100 kDa protein are obtained by 'in vitro' incubation of the 60S and 80S preribosomes. The 100 kDa protein has been purified to homogeneity with the protease activity still associated. The properties of the enzyme are described and its role in the maturation of preribosomes is discussed.

Extranucleolar elements were isolated from mammalian cell nuclei and characterized by correlative biochemical-ultrastructural studies. These complex structures were shown to retain a supra-particle arrangement closely resembling the organizaton in situ of definite nuclear areas. In particular the morphological RNA . protein species, namely perichromatin fibrils and granules, were detected in association with characteristic regions of chromatin. By autoradiographic experiments, we were able to show that perichromatin fibrils represent the morphological state of newly formed heterogeneous nuclear RNA (hnRNA).Chromatin . RNA . protein complexes were further fractionated by means of a treatment dissociating the chromatin component. A minor part of DNA, possibly involved in the biosynthesis and the processing of hnRNA, remains linked to the resulting RNA . protein network.Most of the mechanisms involved in the control of RNA maturation in eukaryotic cells still remain to be elucidated. It has now been clearly established that a class of heterogeneous nuclear RNA (hnRNA) of high molecular weight, rapidly labeled and with a DNA-like composition [l-'51, presents The present study was undertaken in order to establish tentative correlation between biochemical and ultrastructural features of nuclear RNA protein particles and their spatial organization in situ.Abbreviation. hnRNA, heterogeneous nuclear RNA. ~9 1 .We developed an isolation procedure of subnuclear fractions where the ultrastructural organization of definite RNA . protein . chromatin areas appears well preserved. This method could contribute to the characterization of actively transcribed zones in chromatin as well as to a better definition of the events involved in hnRNA metabolism. We here report direct evidence that perichromatin fibrils do indeed represent the morphological state of newly synthesized hnRNA, as previously assumed by autoradiographic studies in situ [18]. MATERIALS AND METHODS Cell Culture and LabelingZadjela Ascitic Hepatoma Cells. Female Wistar rats were inoculated intraperitoneally with approximately 2 x lo7 cells. After 3 days, the cells were collected, washed repeatedly in a phosphate-buffered saline medium (NaC10.12 M, KCl4 mM, Na2HP04 7 mM, KH2P0, 1 mM, NaHCO, 16 mM) and resuspended in Fischer [19] medium (Eurobio), supplemented with 5 % calf serum, 100 U/ml penicillin and 50 pg/ml streptomycin. The cell suspension was then incubated at 37 "C in glass flasks at concentrations varying from 5 x 105/ml to 106/ml according to the experiments planned. Cells were labeled after a 5-h incubation in synthetic medium by addition of radioactive precursors.

Incubation of Chinese Hamster Ovary (CHO) cells for one hour at 43 degrees C results in several obvious changes in protein distribution and protein synthesis. One major protein of the cytoplasm (molecular weight 45,000 daltions), also present as a minor component in the nucleus, rapidly disappeared while several proteins, especially high molecular weight peptides, were induced by heat shock. Localization of the proteins in the cytoplasm, extra-nucleolar chromatin and nucleolar bodies has been carried out. Different sets of induced proteins appear in each subcellular compartment. Four hours after restoration of the normal temperature, the normal pattern of protein synthesis was observed. The 45,000 dalton protein reappeared first. Relations between structural and functional alterations and changes in protein distribution are suggested.

No abstract