The effect of the method of isolation of cell nuclei on the ratios of nuclear proteins to DNA

Dounce, Alexander L.; Ickowicz, Regina

doi:10.1016/0003-9861(69)90408-1

Cited by 29 publications

(7 citation statements)

References 14 publications

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“…Sprague-Dawley rats weighing 180-200g were used for isolation of liver nuclei at pH 5.8, 3.8 and 3.1 and without pH control in the presence of 5mM-CaCl2 (Dounce et al, 1966a;Dounce & Ickowicz, 1969). Calf liver nuclei were isolated at pH3.8 from the liver of freshly slaughtered calves.…”

Section: Materials and Methods Isolation Of Nucleimentioning

confidence: 99%

“…Some of the possible implications of the presence of an extra amount of histone in liver nuclei isolated at pH3.8 or 5.8 or the loss of approximately half the histone complement when liver nuclei are isolated at pH 3.1 or in the presence of CaCl2 without pH adjustment have been discussed previously (Dounce & Ickowicz, 1969;Chanda & Dounce, 1971c;Dounce et al, 1972). We cannot say at the present time how the extra complement of histone is bound in liver nuclei when it occurs, but the observation of Phillips (1968) and Paul & More (1972) that calf thymus chromatin can bind an extra complement of histone when suspended in a solution of free histone may have bearing on this point.…”

Section: Conditions Of Isolation Of Nucleimentioning

confidence: 99%

“…Previous work from this laboratory has indicated that rat liver nuclei isolated either at pH5.8 or 3.8 contain at least twice as much histone as DNA on a dry-weight basis (Umania et al, 1964; Chanda & Dounce, 1971a). On the other hand, when nuclei are isolated at pH 3.1 or in the presence of CaCl2 or MgCl2 without pH control, the ratio of histone to DNA drops to 1.0-1.2 (Dounce et al, 1966a;Dounce & Ickowicz, 1969), although the electrophoretic patterns of histone extracted from nuclei isolated at pH 3.1 or in the presence of CaCl2 or MgCl2 are nearly indistinguishable from those of rat liver nuclei isolated at pH 5.8 or 3.8 (Chanda & Dounce, 1971b).…”

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High total histone/deoxyribonucleic acid ratios for rat liver nuclei

Chanda

Ickowicz

Dounce

1973

Biochemical Journal

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The ratios of total histone to DNA for rat liver nuclei isolated by four methods as well as for calf liver nuclei isolated by one method were determined by obtaining the ratios of the total areas of the electrophoretic histone peaks for the liver nuclei to the corresponding total area given by a known amount of standard calf thymus histone. Ratios of total histone to DNA of approx. 2 for rat liver nuclei isolated at pH3.8 or 5.8 and for calf liver nuclei isolated at pH3.8 were confirmed twice by the above procedure and also by direct measurement, by the method of Lowry et al. (1951), of histone extracted in 0.2m-H(2)SO(4). The histones of calf thymus, calf liver and rat liver were characterized by their amino acid compositions and by polyacrylamide-gel electrophoresis.

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Section: Materials and Methods Isolation Of Nucleimentioning

confidence: 99%

Section: Conditions Of Isolation Of Nucleimentioning

confidence: 99%

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confidence: 99%

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High total histone/deoxyribonucleic acid ratios for rat liver nuclei

Chanda

Ickowicz

Dounce

1973

Biochemical Journal

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“…In their paper, Dounce and Ickowicz (1969) recommended the O· 44M sucrose, pH 5·8, method for the isolation of nuclei when studies on nuclear proteins are to be undertaken. In our experiments we found no advantages in this method and, in fact, the enzyme activities obtained are somewhat lower than other methods.…”

Section: Discussionmentioning

confidence: 99%

“…Since it has been shown that the method of isolation of nuclei has a marked effect on the nuclear protein composition (Dounce and Ickowicz 1969), it was decided to determine the extent to which RNA synthesizing activity was affected. Table 2 shows the results of this series of experiments.…”

Section: Nuclei Preparation Methodsmentioning

confidence: 99%

Characterization of the RNA Synthesizing Activity of Isolated Kidney Nuclei

Cj¹,

Jf²

1975

Aust. Jnl. Of Bio. Sci.

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The limiting factor in RNA synthesis by isolated kidney nuclei is RNA nucleotidyltransferase at high salt concentrations but at low salt concentrations template availability becomes limiting.oc-Amanitin inhibits 85 % of the activity at high salt concentrations but only 20-50 % of the activity at low salt concentrations. Exogenous DNA is utilized at low salt concentrations [up to O·lM (NH4hS041 but not at high salt concentrations. The effect of increasing salt concentration is mainly to cause an increase in the length of chains synthesized. Initiation rates are not increased by high salt concentrations. The apparent Km for UTP is 8-10 11M at high salt concentrations, indicating that assays performed at low UTP concentrations are likely to give inaccurate results. The activation energy for the reaction at low salt concentration is less than that for the reaction at high salt concentration. The RNA synthesizing capacity of kidney nuclei is dependent on the method of isolation, and preparation by a modification of the Chauveau method (Chauveau et al. 1956) yields the most active nuclei.

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Structure and Function of Chromatin

Simpson¹

1973

Advances in Enzymology - And Related Areas of Molecular Biology

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The effect of the method of isolation of cell nuclei on the ratios of nuclear proteins to DNA

Cited by 29 publications

References 14 publications

High total histone/deoxyribonucleic acid ratios for rat liver nuclei

High total histone/deoxyribonucleic acid ratios for rat liver nuclei

Characterization of the RNA Synthesizing Activity of Isolated Kidney Nuclei

Structure and Function of Chromatin

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