Effect of temperature of aldehyde fixation on the radioautographic localization of ribonucleoprotein in nucleoli of HeLa cells. Inhibition by puromycin and actinomycin D.
In efforts to clarify the role of the nucleolus and substructures thereof in the assembly or synthesis of protein associated with formation of the complete ribosome, the effect of variation of some conditions of aldehyde fixation on the intranuclear distribution of lysine-(3)H, arginine-(3)H, and uridine-(3)H was studied by differential grain count in radioautographs of PPLO-free HeLa cells. It was found that the nucleolus is a site of rapid assembly or synthesis of a protein, the synthesis of which is inhibited equally by puromycin (200 microg/ml) and by actinomycin D under conditions inhibitory for ribosomal precursor RNA synthesis (P < 0.01). This protein is fixed by phosphate-buffered formalin or glutaraldehyde at pH 7.3, but the label is diminished by fixation in customarily employed acetic ethanol or in formalin at acid pH. Elevation of temperature of formalin or glutaraldehyde fixatives to 37 degrees C consistently reduces the nucleolar protein label, but not the RNA label, by a proportion identical with that incurred by puromycin or actinomycin inhibition. This proportional reduction of nucleolar protein label occurs without evident loss of total grain count and is independent of length of fixation between 30 min and 4 hr, but it is not observed at 23 degrees C. The data support the interpretation that the proportion of nucleolar protein not fixed at 37 degrees C is associated with nucleolar ribosomal RNA but that it is dissociated at 37 degrees C in formalin or glutaraldehyde fixatives, probably on the basis of ionic dissociation of a conjugated ribonucleoprotein.
D000447:Aldehydes; D000818:Animals; D001120:Arginine / Q000378:metabolism; D001345:Autoradiography; D002466:Cell Nucleolus; D002467:Cell Nucleus; D003593:Cytoplasm; D003609:Dactinomycin / Q000494:pharmacology*; D004792:Enzyme Precursors; D006367:HeLa Cells / Q000032:analysis; D006652:Histological Techniques; D008239:Lysine / Q000378:metabolism; D009698:Nucleoproteins / Q000096:biosynthesis*; D011691:Puromycin / Q000494:pharmacology*; D012270:Ribosomes; D013696:Temperature; D014316:Tritium; D014529:Uridine / Q000378:metabolism
D000447:Aldehydes; D004792:Enzyme Precursors; D009698:Nucleoproteins; D014316:Tritium; D003609:Dactinomycin; D011691:Puromycin; D001120:Arginine; D008239:Lysine; D014529:Uridine