Early nucleolar preribosomal RNA - protein in mammalian cells.

Early steps of ribosomal maturation have been studied by analysis of nucleolar extracts using different extraction procedures. Early 45-S nucleolar RNA is found to be associated with slowly sedimenting elements, distinct from previously described 80-S and 55-S nucleolar preribosomes. This early 45-S RNA has been shown to be of preribosomal type according to the following criteria. (a) When hybridized with nucleolar DNA, competition with rRNA can be observed; (b) its biosynthesis is sensitive to low doses of actinomycin D; (c) it is methylated at an early stage; (d) it contains no linked poly(A) segments. This early 45-S RNA seems to be specifically linked with proteins. The protein content of these early RNA-protein complexes is significantly lower than that for 80-S preribosomes. 45-S RNA sensitivity to nucleolytic activities that can take place in the course of preribosome extraction has been found to be higher in these RNA-protein complexes than in 80-S preribosomes. Identical results were obtained when different mammalian cell species were studied (rat, hamster, or HeLa cells).     

Studies on the molecular arrangement of RNA in tissues with a selective topo-optical reaction of RNA.

Selective demonstration of RNA in tissues was achieved by treating tissue sections with potassium permanganate followed by bisulfite and toluidine blue at pH 1.0 (PBT reaction). It is suggested that this reaction is due to aldehyde groups which are formed by the oxidative cleavage of the pyrimidine rings of RNA which can be selectively demonstrated using bisulfite-toluidine blue as the aldehyde reagent. The specificity of the reaction was tested after RNAase treatment, after acid hydrolysis, and on pure RNA droplets. The aldehyde nature of the reacting groups was checked, after permanganate oxidation, by Schiff's leucofuchsin reagent, and by aldehyde blocking reactions. Two types of intracellular molecular arrangement of RNA molecules could be distinguished by polarization optics after application of the PBT reaction: 1) The strong birefringence, dichroism and metachromatic staining of membrane-bound RNA in ergastoplasm of pancreas, liver and plasma cells indicate a linear (planar) molecular order of RNA molecules on the surface of the membranes, and 2) the isotropic, basophilic staining of RNA not organized in membrane structures (Nissl substance, nucleoli) suggest a random distribution of their dye binding sites.     

Effects of 5-azacytidine on nucleolar RNA and the preribosomal particles in Novikoff hepatoma cells.

Examination of nucleolar RNA from cultured Novikoff hepatoma cells treated for 3 hr with 5 x 10-4 M 5-azacytidine shows that significant amounts of analog-substituted 45S RNA are processed to the 32S RNA species, but 28S RNA formation is completely inhibited. Under these conditions of analog treatment 37% of the cytidine residues in the 45S RNA is replaced by 5-azacytidine. During coelectrophoresis of nucleolar RNA from 5-azacytidine-treated and control cells, the analog-substituted 45S RNA and 32S RNA display reduced mobilities compared to the control 45S RNA and 32S RNA. Coelectrophoresis of analog-substituted and control RNA after formaldehyde denaturation shows no differences in electrophoretic mobility between the two RNA samples, suggesting that 5-azacytidine incorporation may alter the secondary structure of the 45S RNA and the 32S RNA. 5-Azacytidine at 5 x 10-4 M severely inhibits protein synthesis in Novikoff cells by 3 hr. After this length of treatment, however, CsCl buoyant density analysis reveals no difference in density of either the 80S or 55S preribosomal ribonucleoprotein particles when compared to normal particles. Also 5-azacytidine treatment does not appear to cause major changes in the polyacrylamide gel electrophoresis patterns of the proteins in the 80S and 55S preribosomal particles. These results together with previous findings suggest that 5-azacytidine's inhibition of rRNA processing is possibly related to its alteration of the structure of the ribosomal precursor RNAs and is not a consequence of a general inhibition of ribosomal protein formation.     

Studies on the molecular arrangement of RNA in tissues with a selective topo-optical reaction of RNA

Summary Selective, demonstration of RNA in tissues was achieved by treating tissue sections with potassium permanganate followed by bisulfite and toluidine blue at pH. 1.0 (PBT reaction). It is suggested that this reaction is due to aldehyde groups which are formed by the oxidative cleavage of the pyrimidine rings of RNA which can be selectively demonstrated using bisulfite-toluidine blue as the aldehyde reagent.The specificity of the reaction was tested after RNAase treatment, after acid hydrolysis, and on pure RNA droplets. The aldehyde nature of the reacting groups was checked, after permanganate oxidation, by Schiff's leucofuchsin reagent, and by aldehyde blocking reactions.Two types of intracellular molecular arrangement of RNA molecules could be distinguished by polarization optics after application of the PBT reaction: 1) The strong birefringence, dichroism and metachromatic staining of membrane-bound RNA in ergastoplasm of pancreas, liver and plasma cells indicate a linear (planar) molecular order of RNA molecules on the surface of the membranes, and 2) the isotropic, basophilic staining of RNA not organized in membrane structures (Nissl substance, nucleoli) suggest a random distribution of their dye binding sites.     

RNA synthesis in the circular nucleoli of hepatocytes]

By analysis of serial sections it has been revealed that the so-called ring-like nucleoli of hepatocytes consist of a cavity with an amorphous contents surrounded by fibrillar and granular material. Such nucleoli are sometimes encountered in normal animals; the number of ring-like nucleoli increases considerably in chronic pathological process caused by repeated CCl4 injections. The capacity of RNA synthesis in the ring-like nucleoli was revealed by means of electron-microscopic autoradiography.     

Studies on satellite nucleoli in human blastic cells of acute leukaemias.

Blastic cells of acute lymphoid, acute myeloid and acute myelomonocytic leukaemias were studied by means of indirect immunofluorencence to provide more information on the presence of satellite nucleoli in blood cells. According to results, satellite nucleoli were found in a small but constant number of blastic cells disregarding their type and type of acute leukaemia. Satellite nucleoli exhibited a positive immunoreaction for fibrillarin and protein B23 which are characteristic for main nucleolar components. These findings suggest that satellite nucleoli contain fibrillar centers as well as dense fibrillar and granular components or at least proteins characteristic for these nucleolar components. Similarly as in normal and pathological cells of completely different origin, in blastic cells of acute leukaemias the number of satellite nucleoli per cells ranged between 1 and 2.     

Studies on nucleoli of maturing frog erythroblasts

Summary Frog erythroblasts were studied in summer animals with a very active as well as reduced erythropoiesis due to experimental hibernation, the latter being administered in order to get more information on the frequency of various nucleolar types in maturing cells. The results suggest that nucleoli with nucleolonemata are a transitional nucleolar type between compact and ringshaped nucleoli. Since micronucleoli represent final nucleolar maturation changes and compact nucleoli are present in most immature cells, the sequence of nucleolar changes based on the frequency of investigated nucleolar types is as follows: compact nucleolinucleoli with nucleolonemataringshaped nucleolimicronucleoli. The experimental hibernation produces a shift of nucleoli to less active and maturer nucleolar types in all stages of the erythroblastic maturation. In addition, the experimental hibernation produces the formation of ringshaped nucleoli in the first stages of the erythroblastic maturation which in summer animals usually contain compact nucleoli and/or nucleoli with distinct nucleolonemata.