DIFFERENTIATION OF NUCLEOPROTEINS BY INACTIVATION OF PROTEIN-BOUND AMINO GROUPS AND STAINING WITH TOLUIDINE BLUE AND AMMONIUM MOLYBDATE

Abstract

1. A method of staining nucleic acids is described. The method depends upon (a) liberation of the phosphoryl groups of nucleic acids by inactivation of protein-bound amino groups; (b) binding of the phosphoryl groups by toluidine blue; and (c) coupling of some of the bound toluidine blue with polymolybdate. Nucleic acids are stained differently by toluidine blue-molybdate because of variation in the susceptibility of the amino groups of nucleoproteins to inactivation by formaldehyde and nitrous acid. At each stage of inactivation the amount of toluidine blue available to react with molybdate differs for each nucleoprotein. Polymolybdates react with toluidine blue to form a metachromatic complex involving the incorporation of water of dimerization. Hypochromia results when small amounts of this complex are formed at sites of orthochromatic staining by toluidine blue; larger amounts of this complex eclipse coloration by toluidine blue and produce intense metachromasia. 2. By means of the toluidine blue-molybdate stain the deoxyribonucleoprotein and 7 different forms of ribonucleoprotein can be distinguished in the cell. These are: the nucleolus, nucleolinus, parachromatin, perichromosomal, chromosomal, granular and diffuse cytoplasmic ribonucleic acid. The relationship of these observations to known chemical and functional differences in the nucleoproteins of the cell is discussed. 3. A study of seminiferous epithelium, embryonic, and neoplastic cells by the toluidine blue-molybdate method suggests that one type of ribonucleoprotein passes from nucleus to cytoplasm and vice versa during mitosis. Another form, the diffuse cytoplasmic ribonucleic acid, is unaltered during mitosis. It is suggested that these may correspond to the gene-dependent and gene-independent ribonucleic acid, respectively.