IN-SITU FLUORESCENT VISUALIZATION OF NUCLEOLAR ORGANIZER REGION-ASSOCIATED PROTEINS WITH A THIOL REAGENT

Nucleolar organizer regions (NORs) are nucleolus-forming rDNA loops as sociated with argyrophil proteins, the amount of which varies accordin g to the proliferative state of the cell. It has been presumed that th e nucleolar protein-related thiol groups may have a role in selective silver staining. We investigated the nuclear thiol distribution with a fluorescent thiol reagent, coumarinyl-phenyl-maleimide (CPM) in human K-562 myeloblast cultures and found that SH group-related fluorescenc e was brightest in the area of nucleoli, which became highly selective after RNAse digestion. A remarkable co-localization of AgNOR silver r eaction and CPM fluorescence was observed, although occupation of the SH groups by CPM did not prevent the silver staining. We applied the s tain to dual-parameter flow cytometry in combination with DNA content measurements, which provide further information on nucleolar function and changes in experimental and pathological specimens.