Biophysical Studies on the Mechanism of Quinacrine Staining of Chromosomes

Jm, Gottesfeld; Bonner, James; Gk, Radda; Io, Walker

doi:10.1021/bi00711a600

Cited by 47 publications

(13 citation statements)

References 36 publications

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“…In the case of QM, structural changes in chromatin organization can result in a different reciprocal distribution of dye molecules which, in turn, gives rise to different energy transfer efficiency and therefore to different fluorescence emission intensity (Andreoni et al, 1979). In this respect, significant differences in fluorescence intensities of chromosomes stained with QM have been found to be related to the DNA-protein interactions (Gottesfeld et al, 1974). Moreover, the obvious modifications of the QM fluorescence pattern and the decrease in quinacrine fluorescence throughout the GI phase of the 3T3 cell cycle have been also suggested to be due to the structural changes in chromatin and not to the quantity of bound fluorochrome (Moser et al, 1981).…”

Section: Discussionmentioning

confidence: 99%

Fluorescence image cytometry of nuclear DNA content versus chromatin pattern: a comparative study of ten fluorochromes.

Santisteban

Montmasson²,

Giroud³

et al. 1992

J Histochem Cytochem.

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This study is intended to be the first step of an in situ exploration of the intranuclear DNA distribution by image cytomeuy (SAMBA) with several fluorochromes. The nuclear DNA content and the chromatin pattern, revealed by ten fluorochromes (HO, DAPI, MA, CMA3, OM, QM, AO, EB, PI, and 7-AMD), were analyzed on mouse hepatocytes Fied by the Boehm-Sprenger procedure optimal for preserving the chromatin pattem. The question was whether fluotochromes specific to DNA make it possible to accurately quantitate the total nuclear DNA content when the chromatin pattern is preserved. Only HO and MA were found to provide satisfactory quantitation of nuclear DNA content, as assumed by both a small CV and a 4c to 2c ratio equal to 2. PI, EB, 7-AMD, and OM provided higher CV values, although the 4c to 2c ratio was stiU equal to 2. QM, AO, CMA3, and DAPI provided non-reproducible and non-stoichiometric nuclear DNA content measurements under the Fiation conditions used. The intranuclear and the intemuclear SD of the fluorescence intensities describing the fluorescence pattern of the

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Section: Discussionmentioning

confidence: 99%

Fluorescence image cytometry of nuclear DNA content versus chromatin pattern: a comparative study of ten fluorochromes.

Santisteban

Montmasson²,

Giroud³

et al. 1992

J Histochem Cytochem.

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“…Nuclease-resistant segments in transcriptionally inactive chromatin are due to histone-DNA interactions, while the nuclease-resistant segments of active chromatin are due to DNA complexed with both histone and nonhistone proteins. Nuclease-resistant structures of inactive chromatin sediment at 11-13 S and resemble v-bodies (2) in the electron microscope (17 (31)(32)(33) suggest that active chromatin is in a more extended, more DNA-like conformation than inactive chromatin. The electron microscope has revealed differences in the structure of transcriptionally active and inactive regions of chromatin.…”

Section: Discussionmentioning

confidence: 99%

Structure of transcriptionally active chromatin.

Gottesfeld¹,

Murphy²,

Bonner³

1975

Proc. Natl. Acad. Sci. U.S.A.

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108

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Rat-liver chromatin has been fractionated into transcriptionally active and inactive regions [Gottesfeld eta. (1974) Proc. Nat. Acad. Sci. USA 71, 2193USA 71, -2197 and the distribution of nuclease-resistant complexes in these fractions has been investigated. About half of the DNA of both fractions is resistant to attack by the endonuclease DNase IL The nuclease-resistant structures of inactive chromatin are DNA-histone complexes (v-bodies) which sediment at 11-13 S. Template-active chromatin yields two peaks of nucleaseresistant nucleoprotein. These complexes sediment at 14 and 19 S, and contain DNA, RNA, histone, and nonhistone chromosomal proteins. Polyacrylamide gel electrophoresis reveals a complex pattern of chromatin proteins, suggesting that the complexes are heterogeneous in composition. A regular repeating unit in chromatin was first suggested from the x-ray diffraction studies of Pardon et al. (1) MATERIALS AND METHODSChromatin Fractionation. Chromatin was prepared from rat liver by the method of Marushige and Bonner (22) and treated with diisopropylfluorophosphate (DFP) to inhibit endogenous protease activity (23). Fractionation was carried out as diagrammed in Fig. 1; details of this method have been published previously (21).Preparation of Chromatin Subunits. Nuclease-resistant subunits of rat-liver chromatin were prepared as follows:DNase II was added to 10 units per A260 unit of chromatin in 25 mM sodium acetate (pH 6.6). Digestion was carried out at 240 and was terminated after 90 min by raising the pH to 7.5 with 0.1 M Tris-HCl (pH 11). Nuclease-resistant subunits from chromatin fraction P1 were prepared by homogenizing the pellet fraction in 25 mM sodium acetate (pH 6.6) and redigesting with DNase as described above for whole chromatin. Undigested chromatin (about 20% of the input DNA) was removed by centrifugation at 27,000 X g for 10-15 min. The supernatant was layered on isokinetic sucrose gradients in SW25. 1 cellulose nitrate tubes. The gradients were formed according to Noll (24); the parameters were CTOP = 15% (weight/volume), CRES = 34.2% (weight/ volume), and VMIX = 31.4 ml. All solutions contained 10 mM Tris-HCl (pH 8). Centrifugation was at 25,000 rpm for [36][37][38][39][40][41][42] hr. Gradients were analyzed with an ISCO UV Analyzer and chart recorder. Fractions from these gradients were rerun on 5-24% isokinetic sucrose gradients. The parameters were CTOP = 5.1% (weight/volume), CRES = 31.4% (weight/volume) and VMIX = 9.4 ml. Centrifugation was in the SW 41 rotor at 39,000 rpm at 40 for 16.5 hr.Subunits were also prepared from chromatin devoid of histone I. Removal of this histone was accomplished by extraction of Virtis-sheared chromatin (45 V, 90 sec) with 0.5 M NaCl at 4°. The resultant nucleohistone was pelleted by centrifugation in the Ti 50 rotor at 50,000 rpm for 18 hr.The pellet was digested with nuclease as described above.Redigestion of Chromatin Fraction S2. Chromatin of fraction S2 was redigested with nuclease in three different ways: the DNase II present...

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“…DNA was purified from various chromatin fractions as described elsewhere (16). DNA isolated from the S2 chromatin fraction had a single-stranded length of 500 nucleotides, as determined by sedimentation velocity centrifugation under alkaline conditions (17).…”

Section: Methodsmentioning

confidence: 99%