Micro estimation of RNA by the cupric ion catalyzed orcinol reaction

Lin, Robert; Schjeide, O. A.

doi:10.1016/0003-2697(69)90061-x

Cited by 190 publications

(30 citation statements)

References 15 publications

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“…After washing with 0.5 M NH,C1 [l] isoelectric focusing showed that no A-protein was removed from the ribosomes. The protein content of washed ribosomes is 33 & 201, by weight using the microbiuret assay with crystalline insulin as a standard (A::$"' = 1.03 a t 275 nm in 0.01 N HCl) [2] ; RNA was determined by the method of Lin and Schjeide [8] using transfer RNA (General Biochemicals) as a,ptandard and a value of At:$m1 = 22.3 at: 260nm in I 1OmM magnesium acetate and 5 mM Tris-HC1 pH 7.4. The washed ribosomes give a value of = 14.5 a t 260nm in iOmM magnesium acetate and 5mM Tris-HC1 pH 7.…”

Section: Methodsmentioning

confidence: 99%

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50-S Ribosomal Proteins. Purification and Partial Characterization of Two Acidic Proteins, A1 and A2, Isolated from 50-S Ribosomes of Escherichia coli

Möller¹,

Groene²,

Terhorst³

et al. 1972

Eur J Biochem

163

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A method is presented to isolate in pure form two acidic proteins, indicated as A1‐ and A2‐protein from 50‐S ribosome of Escherichia coli. The advantage of this method is that it does not require prior isolation of subunits. The molecular weight of A1 and A2‐protein was found by dodecylsulfate gel electrophoresis to be 11500 with a maximum uncertainty of 1500. Sedimentation equilibrium centrifugation in 5 M guanidine hydrochloride indicates a molecular weight in the range of 12000–13000. The amino acid composition of the two proteins is remarkable and indistinguishable. A‐protein has a high content of alanine and does not contain tyrosine, tryptophan, histidine or cystenie. On gel‐electrofocusing the isoelectric points of A1‐ and A2‐protein are slightly different, namely pH 4.7 and pH 4.85. The amount of A‐protein in actively dividing cells indicates that contrary to 30‐S ribosomes, 50‐S ribosomes have a restricted element of repeat. The number of copies of A‐protein is found to be somewhat larger than two.

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Section: Methodsmentioning

confidence: 99%

“…Catalase (8) 100 000 80 000 60 000 Fig.2A, B). At p H 8.5, a difference in charge is no longer detectable (Fig.…”

Section: Separation and Purity Of A-and A-proteinmentioning

confidence: 99%

50-S Ribosomal Proteins. Purification and Partial Characterization of Two Acidic Proteins, A1 and A2, Isolated from 50-S Ribosomes of Escherichia coli

Möller¹,

Groene²,

Terhorst³

et al. 1972

Eur J Biochem

163

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“…Detection of GSTP1 mRNA by Northern Blot Analysis and GSTP1 Polypeptides by Immunoblot-Total RNA was isolated from Hep3B cells and Hep3B-5-aza-dC clones using an RNeasy RNA isolation kit (Qiagen) and quantified using an orcinol assay (26). Purified RNAs (20 g) were electrophoresed on 1.5% agarose gels in the presence of 2.2 M formaldehyde, transferred to Zeta-Probe ® GT (Bio-Rad) filters, and then assessed for GSTP1 mRNA levels by hybridization with specific 32 P-labeled GSTP1 cDNA probes (ATCC) prepared using Rediprime II DNA labeling system (Amersham Biosciences).…”

Section: Culture Of Hep3b Cells and Treatment With 5-aza-dc And Tsa-mentioning

confidence: 99%

Methyl-CpG Binding Domain Protein 2 Represses Transcription from Hypermethylated π-Class Glutathione S-Transferase Gene Promoters in Hepatocellular Carcinoma Cells

Bakker

Lin

Nelson

2002

Journal of Biological Chemistry

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During the pathogenesis of human hepatocellular carcinoma (HCC), the CpG island encompassing the -class glutathione S-transferase gene (GSTP1) becomes hypermethylated. Repression of transcription accompanying CpG island hypermethylation has been proposed to be mediated by methyl-CpG binding domain (MBD) proteins. We report here that inhibition of transcription from hypermethylated GSTP1 promoters in Hep3B HCC cells, which fail to express GSTP1 mRNA or GSTP1 polypeptides, appears to be mediated by MBD2. Treatment of Hep3B cells with 5-azadeoxycytidine (5-aza-dC), a methyltransferase inhibitor, activated GSTP1 expression, whereas treatment with trichostatin A, a histone deacetylase inhibitor, had little effect. To more precisely assess the contribution of the pattern of GSTP1 CpG island methylation on GSTP1 mRNA expression, Hep3B cells were treated for 72 h with 5-aza-dC and then subjected to limiting dilution cloning. Bisulfite sequencing was used to map the methylation patterns of the GSTP1 promoter region in GSTP1-expressing and -non-expressing clones. In the clone that expressed GSTP1 mRNA determined by Northern blot analysis and quantitative reverse transcriptase (RT)-PCR, widespread demethylation of at least one GSTP1 allele was evident. Chromatin immunoprecipitation experiments revealed the presence of MBD2, but not Sp1, at the GSTP1 promoter in Hep3B cells. In contrast, Sp1 was detected at the GSTP1 promoter in a GSTP1-expressing Hep3B 5-aza-dC subclone. To test whether MBD2 might be responsible for the inhibition of GSTP1 transcription from hypermethylated GSTP1 promoters, siRNAs were used to reduce MBD2 polypeptide levels in Hep3B cells. SssI-catalyzed methylation of GSTP1 promoter sequences resulted in diminished luciferase reporter activity after transfection into Hep3B cells. However, when hypermethylated GSTP1 promoter sequences were transfected into Hep3B cells that had been treated with siRNA-targeting MBD2 mRNA, no repression of luciferase reporter expression was evident. These findings implicate MBD2 in the repression of GSTP1 expression associated with GSTP1 CpG island hypermethylation in HCC cells.

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“…RNA was determined by the orcinol procedure (20). Histones were extracted by 0.4 N H2SO4 and measured by A 28 They were analyzed by electrophoresis on 15% polyacrylamide gels containing 6.25 M urea (21).…”

Section: Chromatinmentioning

confidence: 99%

Chemical and physical properties of fractionated chromatin.

Berkowitz¹,

Doty²

1975

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

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Sonicated chicken reticulocyte chromatin was fractionated into transcriptionally active and transcriptionally repressed components. The active fraction is 8% of the whole chromatin but contains 70% of the newly synthesized chromosomal RNA. This RNA has five times as many hemoglobin RNA sequences as does the RNA in the repressed fraction. The amount of the active fraction in the chromatins of several tissues correlates with their synthetic activity. The molecular weight of the DNA of the repressed fraction is approximately twice that of the active fraction. Moreover, the configuration of repressed chromatin is much more compact, consistent with a much larger sedimentation constant. The transcriptionally active fraction displays a 60 lower melting profile and is highly susceptible to DNase I relative to the repressed fraction. The active fraction contains twice as much non-histone protein and 15% less histone than the repressed fraction and is lacking the lysine-rich and much of tie arginine-rich histones. The fraction of eukaryotic chromatin which is transcriptionally active comprises only a small part of the total genome (1). It is advantageous, therefore, to be able to separate this material from the remainder of the chromatin and to identify how it differs from the much larger part that is transcriptionally inactive. For work in this direction we have chosen 12-day chick embryo reticulocyte chromatin for study, since it is actively transcribing hemoglobin messenger RNA and since it has a transcribable region which comprises a particularly small percentage of the genome and therefore produces a comparatively limited variety of mRNA molecules.Various techniques have been used to fractionate chromatin. The essential break-up of the native chromatin has been accomplished by shearing (2-5), sonication (6), DNase digestion (7), or pressure (5, 8) and the chromatin fragments have been fractionated by differential centrifugation (9), sucrose gradient centrifugation (2, 3, 5), solubility (7), and ion exchange (6) or hydroxyapatite (4) We have sought to obtain a clearer separation of transcriptionally active chromatin (TC) and inactive, repressed chromatin (RC) so that the differences might be more unambiguously assessed. This has been done by combining several fractionation and separation techniques in order to maximize efficient and reproducible separation of a small, discrete portion of the chromatin with transcriptional activity (16). This fraction shows more striking differences in both chemical and physical properties when compared to the remainder of the genome than has generally been reported. MATERIALS AND METHODSChromatin Fractionation and Separation. Blood was collected from 12-day-old chick embryos. The cells were washed in Solution 1 [0.32 M sucrose, 2 mM MgCl2, 1 mM potassium phosphate (pH 6.8)] and then lysed in 10 mM CaCl2. The nuclei were washed in a Dounce homogenizer with a solution containing 0.32 M sucrose, 1 mM MgC12, 1 mM potassium phosphate (pH 6.8), and 0.3% Triton N-101. Nuclei were st...

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Micro estimation of RNA by the cupric ion catalyzed orcinol reaction

Cited by 190 publications

References 15 publications

50-S Ribosomal Proteins. Purification and Partial Characterization of Two Acidic Proteins, A1 and A2, Isolated from 50-S Ribosomes of Escherichia coli

50-S Ribosomal Proteins. Purification and Partial Characterization of Two Acidic Proteins, A1 and A2, Isolated from 50-S Ribosomes of Escherichia coli

Methyl-CpG Binding Domain Protein 2 Represses Transcription from Hypermethylated π-Class Glutathione S-Transferase Gene Promoters in Hepatocellular Carcinoma Cells

Chemical and physical properties of fractionated chromatin.

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