Haemoglobin synthesis in inducible, uninducible and hybrid friend cell clones

Paul, John; Hickey, Ivor

doi:10.1016/0014-4827(74)90522-9

Cited by 43 publications

(11 citation statements)

References 11 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Nuclei were prepared from mouse tissues [29] or mouse-derived Friend 707 cells [33,34] and washed with 1 % (v/v) Triton X-100 to remove nuclear membranes and phosphorylated as previously described [29]. Chromatin was prepared from the nuclei by extracting the nuclei three times with 30 vol.…”

Section: Preparation Of Nuclei and Chromatinmentioning

confidence: 99%

Improved Techniques for the Fractionation of Non-Histone Proteins of Chromatin on Hydroxyapatite

Rickwood

MacGillivray

1975

Eur J Biochem

View full text Add to dashboard Cite

The effect of the dissociation medium on the fractionation of chromatin on hydroxyapatite has been studied. Optimal separations of the histones and non-histone protein are only achieved when columns are run in buffers containing high concentrations of sodium ions. We have modified our previously published method such that the chromosomal proteins can be recovered in virtually quantitative yields. Each of the hydroxyapatite fractions has been analysed with respect to nucleic acid content and the proteins have been analysed by two-dimensional gel electrophoresis.Recent evidence suggests that the non-histone proteins of chromatin play an important role in the control of transcription (see review by MacGillivray and Rickwood [l] [6] after the chromatin has been extracted with dilute acid to remove the basic histones. Alternatively, chromatin has been dissociated in sodium dodecyl sulphate [7] or in various salt solutions [8-201 so that after removal of the DNA by centrifugation [7][8][9]12,13,16,17] gel filtration [9-11,18, 191 or precipitation [14,15,20], the chromatin proteins have been obtained as such [I41 or separated from the histones by ion-exchange chromatography [8,[10][11][12][13][15][16][17][18][19][20] or by electrophoresis [7]. Other workers have used variations or combinations of some of these techniques [21-241. However, some of these methods subject the chromatin to extremes of pH and solvent environment which can be destructive to both proteins [25] and DNA [5,19], while others produce selected fractions [2,3,15] or yield low recoveries of non-histone proteins [ l l , 16,18,19]. Our approach has been to use non-rigorous conditions which yet give good yields of non-histone proteins. In our procedure chromatin is dissociated in 2 M NaCl-5 M urea and applied to a column of hydroxyapatite. This ion-exchange medium is not affected by the presence of high concentrations of urea and the phosphate sites which would otherwise bind basic molecules such as histones are effectively blocked by the sodium ions [26,27]. The acidic proteins bind to the Ca2+ sites of the column and are eluted by increasing the phosphate concentration [26]. Thus in the case of chromatin, we found that the histones are not retained and can be recovered in almost quantitative yield, but only 60%-70% of the adsorbed nonhistone proteins could be recovered using phosphate concentrations up to 0.2 M [28,29]. Further increase in the phosphate concentration merely eluted the DNA from the hydroxyapatite. Recently we have found that solutions of high concentrations of guanidinium hydrochloride to be an efficient solvent for non-histone proteins [30,31]. This communication describes the adaption of our previous procedures to include the use of guanidinium hydrochloride in order to obtain a more efficient recovery of nonhistone proteins. MATERIALS AND METHODS ChemicalsAs far as possible these were of AnalaR grade and were largely obtained from British Drug Houses Ltd (Poole, Dorset, U.K.). AnalaR-grade urea was routinely prepared as an 8 M sto...

show abstract

Section: Preparation Of Nuclei and Chromatinmentioning

confidence: 99%

Improved Techniques for the Fractionation of Non-Histone Proteins of Chromatin on Hydroxyapatite

Rickwood

MacGillivray

1975

Eur J Biochem

View full text Add to dashboard Cite

show abstract

“…MELC appear to be a homogeneous population of erythroid precursor cells (3). Induction of the specific differentiation program characteristic of erythropoiesis includes the synthesis of hemoglobin, the appearance of erythrocyte membrane antigen, the appearance of messenger RNA for globin, and morphological changes similar to those associated with the differentiation of proerythroblasts to orthochromic erythroblasts in bone marrow of normal adult mice (1)(2)(3)(4)(5)(6)(7). NN-Dimethylformamide has been shown to induce MELC to erythroid differentiation (8) in a manner similar to Me2SO.…”

mentioning

confidence: 99%

Induction of erythroid differentiation in murine virus infected eythroleukemia cells by highly polar compounds.

Tanaka¹,

Levy²,

Terada³

et al. 1975

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

194

View full text Add to dashboard Cite

show abstract

“…Fw cells (clone FwT 6/4 1/1) are a thioguanine-resistant subclone derived from an inoculum of C. Friend's culture CIA (9 horse serum or fetal calf serum. Cells from logarithmic-phase cultures were diluted to a concentration of 5 X 104 cells per ml in fresh medium containing Me2SO, butyric acid, or hemin, as specified in the text.…”

Section: Methodsmentioning

confidence: 99%

Globin synthesis and erythroid differentiation in a Friend cell variant deficient in heme synthesis.

Rutherford¹,

Harrison²

1979

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

View full text Add to dashboard Cite

Friend erythroleukemia cells of line Fw are noninducible for hemoglobin synthesis by dimethyl sulfoxide, butyric acid, and other agents. However, these agents were found to induce hemoglobin synthesis if the cells were also treated with exogenous hemin. Butyric acid (or, to a lesser extent, dimethyl sulfoxide) by itself induced accumulation of the erythroid-specific membrane protein spectrin. The basis of the control of globin gene expression by hemin was investigated. Hemin does not control globin and total protein synthesis via the action of the hemin-controlled repressor. Rather, hemin, alone or in combination with dimethyl sulfoxide, induces accumulation of globin mRNA and nuclear globin RNA sequences. horse serum or fetal calf serum. Cells from logarithmic-phase cultures were diluted to a concentration of 5 X 104 cells per ml in fresh medium containing Me2SO, butyric acid, or hemin, as specified in the text.Determination of the Rate of Globin Synthesis. Cells were resuspended in F12 medium without leucine but containing 15% dialyzed horse serum, and containing hemin where appropriate, and were labeled for 15 min with 20,Ci (1 Ci = 3.7 X 101. becquerels) of [3H]leucine per ml. Cells were washed, lysed for 15 min in 0.5% Nonidet P40 in buffer containing 0.81% NaCl, 0.03% Mg acetate, and 0.12% Tris-HCl, pH 7.4. The nuclei were removed by centrifugation at 400 X g for 15 min and the supernatant was precipitated at -20°C in acid acetone (2% vol/vol concentrated HCO in acetone), with 10-20 mg of DBA/2J or Porton Swiss mouse hemoglobin added as carrier. Globin chains were separated on CM-cellulose (Whatman CM-52) columns in 8 M urea buffer containing mercaptoethanol (10). A gradient of 0.017-0.050 M Na2HPO4, pH 6.8, was used. The column eluates were monitored for absorbance at 280 nm and a sample from each fraction was assayed for radioactivity.Preparation of Cytoplasmic RNA. Cells were washed and lysed in Nonidet P40, and nuclei were removed as above. Cytoplasmic RNA was then extracted (11).Preparation of Nuclear RNA. Nuclei were purified from whole cells by the citric acid method (12). Nuclear RNA was extracted by a modification of the method of Glisin et al. (13) in which nuclei suspended in sodium dodecyl sarcosinate are treated with proteinase K before centrifugation in cesium chloride.Determination of Globin-Specifying Sequences in RNA. The proportions of globin-specifying sequences in total cytoplasmic RNA and total nuclear RNA were determined by titration hybridization (11,14) The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

show abstract

Haemoglobin synthesis in inducible, uninducible and hybrid friend cell clones

Cited by 43 publications

References 11 publications

Improved Techniques for the Fractionation of Non-Histone Proteins of Chromatin on Hydroxyapatite

Improved Techniques for the Fractionation of Non-Histone Proteins of Chromatin on Hydroxyapatite

Induction of erythroid differentiation in murine virus infected eythroleukemia cells by highly polar compounds.

Globin synthesis and erythroid differentiation in a Friend cell variant deficient in heme synthesis.

Contact Info

Product

Resources

About