Electron Microscopic and Biochemical Characteristics of Nuclei and Nucleoli Isolated From Rat Liver

Abstract

Rat liver nuclei were freed of cytoplasmic contamination by washing with Triton-X-100 and subsequent centrifugation through 2.2 M sucrose. Electron microscopic examination showed that the outer membranes of the nuclei had been removed, but that the nuclei otherwise resembled the nuclei of intact liver. Morphological studies, chemical estimations of DNA, RNA, and protein and the estimation of cytoplasmic "marker" enzymes suggested that contamination of nuclei by cytoplasmic components was limited. These nuclei were obtained in yields of about 70% and were suitable for the isolation of nucleoli. Nucleoli were isolated by the breaking of the nuclei by ultrasound and subsequent differential centrifugation. In ultrastructural appearance, the isolated nucleoli resembled nucleoli in intact tissue. However, at high magnifications the "granular" component of isolated nucleoli appeared to consist of tightly twisted fibers. The nucleoli could be obtained in yields of at least 30%, and the values for the chemical composition of the isolated nucleoli agreed with values previously reported.

On the Attachment of the Nuclear Pore Complex

“…It is well to note that the nuclear pores should indeed be absent, since the nuclear membranes, which define the pores, are unequivocally absent. The absence of pore complexes from earlier electron micrographs is most likely a function of fixation conditions, since we and others (46,47) clearly see well-defined structures. In fact, detergent treatment of the nuclei promotes recognition of the pore complexes by removal of the obscuring membranes.…”

“…1 a). Furthermore, many reports indicate that gross morphology is unaltered, except for removal of the outer nuclear membrane during isolation of nuclei by detergent solubilization of cytomembranes (27,(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)) (see Fig. 1 b).…”

Isolation of Two Distinct Classes of Polysomes From a Nuclear Fraction of Rat Liver

“…Uniformly labeled amino acid-'4C mixture and orotic-6- 4 The determinations of RNA, DNA, and protein have been described previously (21).…”

“…ELECTRON MICROSCOPY: The procedures for fixation, embedding, sectioning and staining have been described (21). Ribosomal preparations were placed directly on Formvar grids for examination with the electron microscope.…”

“…In the present study, we have washed isolated liver nuclei in rat liver cell sap containing Triton-X-100. This detergent has been found to remove the outer nuclear membrane and its attached ribosomes, leaving the interior of the nucleus morphologically intact (21). This treatment liberated a fraction of undegraded polysomes which we have called "outer membrane polysomes."…”