Isolation of nucleoli from rat liver in the presence of magnesium ions

doi:10.1016/0014-4827(72)90264-9

Experimental Cell Research

Volume 71, Issue 1, March 1972, Pages 65-74

https://doi.org/10.1016/0014-4827(72)90264-9 Get rights and content

Abstract

A procedure for the isolation of nucleoli from rat liver in the presence of Mg²⁺ ions was developed. Addition of 0.05 mM MgCl₂ to the 0.34 M sucrose in which isolated nuclei were suspended for sonication was found to protect nucleoli when nuclei prepared with 2.3 M sucrose containing 10 mM MgCl₂ were disrupted completely by sonication. The nucleolar preparation possesses almost identical properties with those of nucleoli prepared by the conventional Ca²⁺-method in the following points: (1) morphological characteristics as revealed by light and electron microscopy; (2) $RNA DNA$ ratio of around 1.3; (3) sedimentation profiles and labeling patterns of nucleolar RNA; (4) polyacrylamide gel electrophoresis of acetic acid soluble proteins. Furthermore, Mg²⁺-nucleoli showed a higher RNA synthetic activity than Ca²⁺-nucleoli. In addition, this method has proved to be advantageous in the case of regenerating liver in which nuclei and nucleoli are somewhat more fragile to sonication.

References (21)

H Busch et al.
Exptl cell res
(1967)
PE Spahr
J mol biol
(1962)
PL Ballard et al.
J biol chem
(1966)
OH Lowry et al.
J biol chem
(1951)
M Muramatsu et al.
Biochim biophys acta
(1966)
M Izawa et al.
Biochim biophys acta
(1969)
M Muramatsu et al.
J biol chem
(1965)
M Muramatsu et al.
Cancer res
(1963)
SB Weiss
M Dixon et al.
The enzymes

There are more references available in the full text version of this article.

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^☆: This work was supported in part by grants from the Ministry of Education of Japan.

^∗: Present address: Mitsubishi-Kasei Institute of Life Sciences, c/o Central Research Laboratory, Mitsubishi Chemical Industries Limited, 290 Hisamotokamoi-cho, Kawasaki, Japan,

^∗∗: Present address: Department of Biochemistry, Tokushima University School of Medicine, Tokushima, Japan.

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Isolation of nucleoli from rat liver in the presence of magnesium ions☆

Abstract

Exptl cell res

J mol biol

J biol chem

J biol chem

Biochim biophys acta

Biochim biophys acta

J biol chem

Cancer res

The enzymes