Two chromatin fractions with different metabolic properties of non-histone proteins and of newly synthesized RNA.

Digestion of chromatin with micrococcal nuclease under mild conditions results in the release of a minor chromatin fraction showing an increased RNA and non-histone protein content, a fast turnover of the non-histone proteins and the presence of rapidly labelled heterogeneous nuclear RNA (hnRNA) with half-life of about 20 min. Further digestion of the chromatin leads to the elimination of about 19% of the initial chromosomal DNA, thus leaving a second chromatin fraction relatively resistant to nuclease attack. This fraction has a low protein and RNA content and contains only metabolically stable non-histone proteins. No differences in the histone complement of the two fractions was found except for a 40% deficiency of H1 in the minor fraction.     

Purification and properties of tyrosinases from Vibrio tyrosinaticus

Rat liver chromatin which has been briefly sonicated is fractionated by treatment with low concentrations of magnesium ion. At 1.5 mm Mg²⁺, where approximately 20–25% of the chromatin remains soluble after low-speed centrifugation, chemical and physical analysis of the Mg-soluble and Mg-insoluble chromatin fractions show that the fractions possess markedly different properties. The Mg-soluble chromatin has more protein and RNA than the Mg-insoluble chromatin. The histone composition of the two fractions as shown by electrophoretic analysis is similar, but many of the acidic proteins are qualitatively and quantitatively different. The molecular weight of the Mg-soluble chromatin is less than that of the insoluble chromatin based on sedimentation behavior and gel filtration experiments. The soluble chromatin has nearly twice the template activity for RNA synthesis in vitro with added RNA polymerase as the Mg-insoluble chromatin and contains approximately 80% of the in vivo rapidly labeled RNA found in the total chromatin preparation. In addition the Mg-soluble chromatin has a significantly greater amount of “accessible” DNA (62%) as measured by polylysine binding than Mg-insoluble chromatin (48%). The data suggest that (a) fractionation of chromatin preparations can be achieved by titration with Mg²⁺, and (b) chromatin soluble in low concentrations of Mg²⁺ may be enriched in actively transcribed portions of the genome.     

Isolation from Friend erythroleukemia cells of an RNase-sensitive nuclear matrix fibril fraction containing hnRNA and snRNA

Chromatin-depleted nuclei (CDN) were prepared from Friend erythroleukemia cell nuclei by partial digestion with DNase I and extraction of the chromatin by 2 mM EDTA as described in the preceding paper (Long and Ochs, 1983. Biol. Cell 48, 99-108). These structures contained dense networks of matrix fibrils surrounded by distinct laminae but no morphologically distinct residual nucleoli. CDN disrupted by gentle shearing or 1 microgram/ml RNase were fractionated into laminae and matrix fibrils by differential centrifugation. Protein composition of the lamina fraction was dominated by two prominent lamina proteins that were not detectable in the matrix fraction. Mild RNase treatment led to a conversion of the fibrous network to a particulate morphology while mild shearing resulted in an apparently unaltered fibril fraction. The matrix fibril fractions contained hnRNP proteins and the snRNAs. These results suggest that EDTA-prepared CDN may provide a system for studying snRNP-hnRNP interactions and hnRNP processing that is less complex than intact nuclei.     

Appearance of rapidly labeled, high molecular weight RNA in nuclear ribonucleoprotein. Release from chromatin and association with protein.

Chromatin and nuclear ribonucleoprotein (nRNP) have been prepared from a human carcinoma cell line. Following a 1-hour (3H)uridine pulse, 60 to 70% of the nuclear radioactivity, after removal of nucleoli, was found in the chromatin, the balance in nRNP. This was true whether the chromatin and nRNP were separated by velocity centrifugation or by isopycnic centrifugation on Metrizamide gradients. Radioactivity in chromatin and nRNP was found in high molecular weight RNA, with mean sedimentation coefficients of 20 S and 15 S, respectively, as determined on sodium dodecyl sulfate-sucrose gradients. Experiments on the kinetics of appearance of radioactivity in the RNA of the two fractions suggest that some of the chromatin-associated RNA is precursor to nRNP-RNA. The proteins of nRNP are complex as revealed by sodium dodecyl sulfate gel electrophoresis. The contamination by chromatin protein was estimated to be 5%. Experiments involving short pulses of (3H)tryptophan, and pulse-chase, suggested that the rapidly turning over proteins of nRNP were not complexed with RNA while still associated with chromatin. However, it was also shown that the radioactivity in nRNP following short pulses of (3H)tryptophan did not correspond to the major bands seen on stained sodium dodecyl sulfate gels. It is therefore concluded that the protein of nRNP consists of two classes: species present in large amounts, possibly common to all RNA in nRNP, which are relatively stable and may be complexed to RNA still associated with chromatin; and a large number of rapidly turning over species, each present in small amounts and associated with nRNP only after its release from chromatin.     

大鼠衰老过程中肝细胞核及染色质的体外转录活性

用同位素掺入法研究不同年龄大鼠的肝细胞核及染色质体外转录活性,所得结果表明:(1)老年大鼠肝细胞核的转录起始能力较断乳鼠及青年鼠分别下降68％及56％。(2)大鼠肝细胞核内与染色质结合的RNA聚合酶所致的转录活性随增龄呈近似线性下降,而不与染色质结合的RNA聚合酶所致的转录活性随增龄则无变化。(3)老年大鼠肝染色质体外转录活性较断乳鼠及青年鼠分别下降52％及35％。这些结果提示。老年大鼠肝染色质功能的改变可能是转录活性改变的主要原因。     

The distribution of ribosomal ribonucleic acids among subcellular fractions from bacteria and the adverse effect of the membrane fraction on the stability of ribosomes

1. The distributions of nucleic acids and protein among fractions obtained by differential centrifugation from species of Pseudomonas, Aerobacter, Escherichia, Proteus and Bacillus have been studied. 2. The DNA in a cell wall-membrane fraction obtained by low-speed centrifugation from the Gram-negative species could be removed by homogenizing and subsequent washing. About 7-14% of the total RNA remained firmly attached and resembled ribosomal RNA in base composition. A similar fraction from the Gram-positive B. subtilis contained about one-half of the total bacterial DNA and only 60% of this could be removed by homogenizing and subsequent washing. 3. A deposit obtained by high-speed centrifugation could be separated into a heavy ribosome layer and a light turbid layer. In E. coli B the latter contained about equal concentrations of RNA and DNA and accounted for about one-half of the total bacterial NADP-activated 6-phosphogluconate dehydrogenase. 4. The washed cell wall-membrane fraction from most species accelerated the degradation of ribosomes. In Pr. vulgaris the activity of this fraction was exceptionally high and resulted in the progressive degradation of ribosomes during their isolation from this species. 5. A possible connexion between ribosome degradation and the synthesis of flagella is discussed in the light of these results.     

Binding of benzo(a)pyrene to rat liver nuclear matrix

Binding of benzo(a)pyrene (B(a)P) to nuclei isolated from rat liver was investigated. After incubation with ¹⁴C-B(a)P, the nuclei were subfractionated into an envelope fraction, two chromatin fractions and a matrix fraction. About 50% of the B(a)P that entered the nuclei was associated with the matrix fraction. Covalently bound B(a)P in the matrix fraction also exceeded that in the chromatin fractions. The radioactivity of ¹⁴C-B(a)P attained by the matrix DNA was 3–5 times higher than that attained by the chromatin DNAs. These findings suggest that the nuclear matrix is a major intranuclear binding site of B(a)P.     

Chromatin-nuclear envelope complex from rat liver: isolation and purification

We describe a method for the isolation of a fraction of nuclear envelope (NE) from rat liver. The method includes mild treatment of pure nuclei with either endonuclease of DNase I under low ionic strength conditions in the presence of magnesium, which allows the nucleomeric organization of the chromatin (Ch) to be preserved. The NEs were purified by centrifugation in sucrose gradients followed by floatation in sucrose. No more than 3% of the Ch present in the purified Ch-NE complexes was due to the non specific adsorption of Ch to the NE. The main components of the complex (Ch and NE) retained their in situ ultrastructure. The complex consisted of 9--10% DNA, 3--4% RNA, about 63% protein and about 24% phospholipids.     

RNA synthesis in isolated HeLa cell nuclei.

RNA synthesis has been studied in isolated nuclei of HeLa cells. The incubation medium has been optimized for RNA synthesis and the requirements for the presence of specific components previously used by other investigators has been examined. Nuclei isolated by centrifugation through 2 M sucrose synthesize RNA linearly for at least 1 h only at low temperature (25 degrees C). Low molecular weight RNA is found in the supernatant fraction after incubation; this RNA accounts for about 10% of the RNA synthesized. The RNA which remains within nuclei is of high molecular weight and processing of this RNA into molecules of the size of cytoplasmic mRNA does not seem to occur in isolated nuclei. We have studied the effect of an inhibitor of protein-nucleic acid interaction - aurintricarboxylic acid - on RNA synthesis by isolated nuclei. At concentrations below 0.1 mM, this drug does not inhibit RNA synthesis effectively, whereas at concentrations above 0.1 mM it inhibits RNA synthesis by about 80%. In view of the proposed mechanism of action of aurintricarboxylic acid, we suggest that completion of nucleotide chains initiated before nuclei isolation accounts for 20% of the RNA synthesized in our system by isolated nuclei, whereas nucleotide chains initiated during the in vitro incubation account for 80% of the RNA synthesized.     

Fractionation of chromatin by differential solubility in dilute salt.

Chromatin prepared from the livers of rats was fractionated on the basis of solubility in dilute NaCl. Neither of the fractions obtained was enriched in newly synthesized DNA. The salt-soluble fraction had a higher protein content (usually up to 50%) relative to the DNA, and contained 72% or more of the rapidly synthesized RNA. This RNA was found to be complexed with the salt-soluble deoxyribonucleoprotein, not merely co-solubilized with it. Also, polylysine-binding studies showed that about 70% or more of the nucleic acid phosphates were accessible as compared to about 40% in the unfractionated chromatin. These properties suggested that the soluble fraction was enriched in activity transcribed chromatin. In contrast molecular hybridization studies showed that the complexity of the DNA and its homology with cDNA transcribed from rat-liver polysomal mRNA were the same as those of DNA from unfractionated chromatin, or from the salt-insoluble fraction. This suggests that the criteria commonly accepted as distinguishing between euchromatin and heterochromatin in vitro are not invariably valid.     

Enrichment for the globin coding region in a chromatin fraction from chick reticulocytes by endonuclease digestion.

A nuclease-sensitive fraction was obtained from chick reticulocyte chromatin by brief digestion with an endonuclease (DNAase II, deoxyribonucleate 3'-oligonucleotidohydrolase, EC 3.1.4.6). The nuclease-sensitive fraction typically contained less than 1% of the chromatin-DNA but about 50% or more of the nascent chromatin-bound RNA. Hybridization of chick globin complementary DNA to the DNA component of the nuclease-sensitive fraction of reticulocyte chromatin indicated a 3--5 fold enrichment for the globin coding region of the chromatin. The control experiment utilizing DNA from a nuclease-sensitive fraction of chick liver chromatin did not show a comparable enrichment for the globin coding region. This suggests that the endonuclease-effected enrichment for the globin coding region in the nuclease-sensitive fraction of reticulocyte chromatin is to some degree specific for structural genes transcribed in reticulocytes.