Separation and characterization of transcriptionally active and inactive nuclear subfractions of AKR mouse embryo cells

R. A. Webster, H. L. Moses, T. C. Spelsberg

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

This study was initiated with the objective of separating and characterizing two or more nuclear subfractions, which could then be compared with respect to their relative propensity for binding carcinogenic polycyclic aromatic hydrocarbons. Nuclei were isolated from cloned AKR 2B mouse embryo cells, which are susceptible to transformation by chemical carcinogens. The nuclei were mechanically sheared and subfractions were separated by sedimentation through a 0.17 to 1.7 M sucrose gradient. When the cells were treated with [3H] uridine for 30 min, most of the label incorporated into RNA was recovered in the top region of the gradients, which represented Nuclear Subfraction I. The majority of the chromatin DNA, however, was localized in the bottom region (Subfraction II) and the pellet (Subfraction III). Precipitation (with CaCl2) of the rapidly labeled RNA of Subfraction I along with the chromatin DNA suggested that the label was present in nascent RNA chains still attached to the chromatin. Thus, the transcriptionally active chromatin seemed to be localized in Nuclear Subfraction I. The chromatin of subfraction I was also the best template for RNA synthesis in vitro with exogenous bacterial polymerase. The protein and RNA content of Subfraction I was greater than that of the other two subfractions and whole chromatin. Electron microscopy revealed the presence of membrane material in Subfractions I and II, with little such material in Subfraction III. Subfraction I differed from Subfractions II and III and whole chromatin with respect to thermal denaturation of the DNA and histone composition (as determined by gel electrophoresis). The acidic protein composition (as determined by gel electrophoresis) differed for the chromatin of all three nuclear subfractions.

Original languageEnglish (US)
Pages (from-to)2896-2904
Number of pages9
JournalCancer Research
Volume36
Issue number8
StatePublished - 1976

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Inbred AKR Mouse
Chromatin
Embryonic Structures
RNA
Electrophoresis
Gels
Nucleic Acid Denaturation
Uridine
DNA
Polycyclic Aromatic Hydrocarbons
Carcinogens
Histones
Sucrose
Electron Microscopy
Proteins
Hot Temperature
Membranes

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

Separation and characterization of transcriptionally active and inactive nuclear subfractions of AKR mouse embryo cells. / Webster, R. A.; Moses, H. L.; Spelsberg, T. C.

In: Cancer Research, Vol. 36, No. 8, 1976, p. 2896-2904.

Research output: Contribution to journalArticle

Webster, R. A. ; Moses, H. L. ; Spelsberg, T. C. / Separation and characterization of transcriptionally active and inactive nuclear subfractions of AKR mouse embryo cells. In: Cancer Research. 1976 ; Vol. 36, No. 8. pp. 2896-2904.
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