Non-histone proteins in fractionated chromatin before and after DNAase II treatment

Rakowicz-Szulczynska, Ewa M.; Horst, Antoni

doi:10.1016/0005-2787(81)90105-2

Cited by 13 publications

(3 citation statements)

References 19 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Other investigators have repeatedly shown that the immunological specificity of chromosomal proteins depends on the presence of proteins that bind tightly to DNA (34)(35)(36)(37). The 2 M NaCl resistant chromatin fraction has been shown to be more sensitive to digestion with DNase II than native chromatin and to be more enriched in active chromatin (38). Perle et al (39) reported that some developmentally regulated nonhistone proteins were found to be both tightly bound to DNA and localized near DNase sensitive domains of precartilage cell chromatin.…”

Section: Resultsmentioning

confidence: 99%

Nuclear matrix DNA from chicken erythrocytes contains beta-globin gene sequences.

Hentzen¹,

Rho²,

Bekhor³

1984

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

View full text Add to dashboard Cite

Nuclear matrices containing residual DNA were isolated from chicken erythrocytes after extraction of purified nuclei with buffered 2 M NaCI. After further purification of this residual DNA, it was found to contain high concentrations of -Wglobin gene sequences as assayed by dot hybridization with 32P-labeled nick-translated pHB1001. Electron microscopy of a random sample of this residual DNA fraction shows the DNA to be intimately associated with protein at various intervals. A hypothesis for enrichment of active genes in residual DNA from purified chromatin or in nuclear matrix DNA is also discussed.During the past decade many diverse experimental strategies have been applied to the study of eukaryotic transcriptional regulation by the nonhistone chromosomal proteins (1). In spite of all of these efforts, the nonhistones presumably responsible for tissue-specific transcription remain elusive. Studies on DNA sequences have provided evidence suggesting that the nucleotide sequences offlanking and intervening sequences are essential for correct gene expression (2). Nevertheless, analysis of restriction enzyme digests of chicken DNA for ,-globin (3), ovalbumin (4), conalbumin (5), and lysozyme (6) have shown that these genes appear to be indistinguishable between tissues where the genes are either active or inactive. Furthermore, comparison of the sequences around the putative promoter sites of the ovalbumin gene (7) cloned from the DNA of producer and nonproducer tissues revealed no differences. Thus it is reasonable to suggest that the nucleotide sequence alone is insufficient for genomic activation of tissue-specific transcription. Although gene-specific nonhistone proteins have not yet been found in chromatin obtained from specialized tissues, results supporting that possibility have been reported (8)(9)(10)(11)(12)(13).Of the total nuclear proteins, the insoluble nonhistones are significant in their content of tissue-specific proteins (14), in the structure of the nuclear matrix (15), and in their association with transcriptionally active genes (16). Recently, studies by Pettijohn's group also suggested that an insoluble nonhistone protein of high molecular weight appears to be specific to cells undergoing active proliferation (17). Previous results from our laboratory have shown that it is possible to isolate from purified chromatin a DNA fraction significantly enriched in tissue-specific gene sequences (18)(19)(20). We suspect that this DNA, which we have designated DNA-P, is similar to that DNA found associated with the nuclear matrix. Transcribed sequences are associated with the nuclear matrix (21), and these sequences may form the base of the DNA loops in which they reside (22). We show by dot hybridization that the insoluble DNA fraction isolated from chicken erythrocyte nuclear matrices contains high concentrations of f3-globin gene sequences, although this gene is known to be inactive in mature chicken erythrocytes (23). MATERIALS AND METHODSIsolation of DNA Associated with Nonhistones from th...

show abstract

Section: Resultsmentioning

confidence: 99%

Nuclear matrix DNA from chicken erythrocytes contains beta-globin gene sequences.

Hentzen¹,

Rho²,

Bekhor³

1984

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

View full text Add to dashboard Cite

show abstract

“…Chromatin was pelleted at the bottom of the tube, nucleoplasm (residual fraction after extraction with 0.14 M NaCl) was recovered from the top, and nuclear membranes were taken at the interface. Chromatin was further fractionated by extraction with 0.35 M and 2 M NaCl as described (25 Solid-Phase Radioimmunoassay. Chromatin was resuspended in 1 mM Tris Cl/1 mM EDTA at pH 7.4 and adsorbed to the wells of polyvinyl chloride microwell plates at 4°C, as described (27), but without chromatin sonication.…”

mentioning

confidence: 99%

Chromatin binding of epidermal growth factor, nerve growth factor, and platelet-derived growth factor in cells bearing the appropriate surface receptors.

Rakowicz-Szulczynska¹,

Rodeck²,

Herlyn³

et al. 1986

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

170

View full text Add to dashboard Cite

We analyzed the uptake and intracellular distribution of 12-5-labeled epidermal growth factor, nerve growth factor, and platelet-derived growth factor in different cell lines that express or do not express the respective surface receptors for these factors. After 1 hr of incubation, all three growth factors were detected in the cytoplasmic fraction and in the nucleus, tightly bound to chromatin. The amount of chromatin-bound growth factors continued to increase during the incubation, and analysis at 48 hr revealed each chromatinbound labeled growth factor in a nondegraded form. After limited digestion of chromatin with DNase 1 (10-20% digested sequences), specific release of all three growth factors was detected only after 1 hr of incubation but not after 24 and 48 hr, suggesting that the DNA regions involved in growth factor binding became nuclease-resistant. Binding of labeled epidermal growth factor and nerve growth factor to isolated chromatin was inhibited by monoclonal antibodies specific for the respective growth factor receptor. The data suggest that chromatin binding may represent an important step in the pathway of growth factor action.The precise mechanism by which different growth factors exert their effects is unclear, although interaction with the appropriate surface receptor and internalization (1-9) are common to all of these factors. The fate of internalized growth factors is controversial. Nerve growth factor (NGF)

show abstract

“…Chromatin was isolated as described by Rakowicz-Szulczynska et al (1981). Briefly, the purified nuclei were washed with 10 mM Tris/HCl, pH 8.3, 140 mM NaCl.…”

Section: Cell Fractionation Of Mammary Gland Tissuementioning

confidence: 99%

A mammary‐derived growth inhibitor (MDGI) related 70 kDa antigen identified in nuclei of mammary epithelial cells

Müller

Kurtz

Vogel

et al. 1989

Journal Cellular Physiology

View full text Add to dashboard Cite

The aim of the present study was to investigate the expression of the mammary-derived growth inhibitor (MDGI) and the subcellular localization of MDGI-related antigens in bovine mammary glands. Cell-free translation of poly(A+) = RNA, immunoprecipitation with rabbit anti-MDGI-antibodies, and estimation of the relative contents of MDGI by a radioimmunoassay in mammary tissue of different functional states revealed that the 13 kDa MDGI was dramatically increased in terminally differentiated mammary tissue compared with the proliferating tissue from pregnant animals. To address the question of tissue localization, polyclonal anti-MDGI antibodies and antibodies directed against a synthetic peptide corresponding to residues 69 to 78 of MDGI were used. Western blotting of tissue fractions revealed the cytosolic and microsomal localization of MDGI. Additionally, both types of antibodies detected a 70-kDa antigen in the nuclear fraction of differentiated mammary glands. Salt extraction and DNase I digestion of isolated nuclei, as well as chromatin purification, indicated an association of the 70-kDa antigen with the chromatin. By means of the immunogold technique, MDGI-related antigens were localized within euchromatic nuclear regions of epithelial cells in the intact differentiated mammary gland. The immunostaining was markedly diminished in the proliferating tissue. This finding raises the possibility that MDGI and the 70-kDa antigen influence cell proliferation by acting on gene expression within the nuclei of mammary glands.

show abstract

Non-histone proteins in fractionated chromatin before and after DNAase II treatment

Cited by 13 publications

References 19 publications

Nuclear matrix DNA from chicken erythrocytes contains beta-globin gene sequences.

Nuclear matrix DNA from chicken erythrocytes contains beta-globin gene sequences.

Chromatin binding of epidermal growth factor, nerve growth factor, and platelet-derived growth factor in cells bearing the appropriate surface receptors.

A mammary‐derived growth inhibitor (MDGI) related 70 kDa antigen identified in nuclei of mammary epithelial cells

Contact Info

Product

Resources

About