A Cytochemical and Radioautographic Study of Human Tissue Culture Cell Nucleoli

Abstract

Fine structural aspects of human tissue culture cell nucleoli were studied by cytochemical and radioautographic methods. Ribonuclease and pepsin digestions were carried out on glutaraldehyde-fixed cells that, in some instances, were labeled with thymidine-~H prior to digestion. Double digestion by ribonuclease and pepsin revealed a fine fibrillar reticulum that appears to be the supportive structure of nucleolonemal threads. The nature of the reticulum remains to be determined. The question of whether it may represent a dispersed form of chromatin was raised. Structural findings suggested such an hypothesis but the results of radioautographic studies do not support it. The rcticulum showed a striking absence of radioactive labeling following a 3 hr incorporation of thymidine-3H. Only few silver grains were observed occasionally in the fibrillar nucleolonema that may or may not be significant. The radioautographic results are believed to be inconclusive for the various masons discussed. The possibility that the reticulum is composed of proteins has to be considered. It appears that basic proteins can resist pepsin digestion in aldehyde-fixed cells. Individual chromatin fibrils were found to be associated with the nucleolar reticulum. It is possible that these alone represent the dispersed genetically active chromatin of nucleoli.

Fractionation of the nuclear matrix. I. Partial separation into matrix protein fibrils and a residual ribonucleoprotein fraction.

“…The presence of polypeptides of similar molecular weight as major constituents of a variety of isolated nuclear fractions (1,4,10,17,32,48) suggests a fundamental structural role for these nonhistone proteins. In this regard, a number of in situ observations have indicated proteinaceous fibrous struc-tures, generally 30-100 fl in width, associated with or in close proximity to a variety of intranuclear structures including interchromatinic and perichromatinic fibers and granules (12,42,50), nuclear pore complexes (34,36,38,53), nucleoli (46,47), and in situ observed matrix structures (15,26). Protein fibrils of similar dimensions and staining characteristics are a major structural component of the isolated nuclear matrix .…”