Proteinase activity of nuclei from various tissues towards endogenous histones]

The proteinase activities of nuclei isolated from tissues differing in their mitotic activities (brain, thymus, liver, ascite lymphoma) towards the histones and non-histone acid -- extractable proteins were studied. The sensitivity of different histone fractions to nuclear proteinase depends on temperature and time of nuclei incubation under conditions providing for complete dissociation of chromatin proteins from DNA (2 M NaCl--5 M urea). The proteinase activity in the brain and thymus nuclei is revealed only under prolonged (43 hrs) incubation of the nuclei at 25 degrees C, which is accompanied by partial proteolysis of histone H1. Histone H4 from brain nuclei and histone H2a from thymus nuclei are preferably degraded. In the nuclei isolated from the mice ascite cell lymphoma NK/ly and from rat liver the enzyme activity is revealed mainly towards the arginine-enriched histones H3 and H4. The proteolysis of the arginine-enriched histones in tumour cell nuclei is more complete. A high sensitivity to proteolysis was observed for non-histone acid-extractable proteins with low electrophoretic mobility, which were found in brain and tumour cell nuclei.     

Phospholipids in plant and animal chromatin

Isolated hepatic nuclei and hepatic chromatin have been analysed for their DNA, RNA, protein and phospholipid content. The protein/DNA ratio is 3 for nuclei and 1.95 for chromatin extracted from Triton X-100 treated nuclei. The phospholipids, (2.36 +/- 0.91 (S.D.) per cent of the total nuclear material), are lost during the chromatin preparation mainly during the Triton X-100 washings of the nuclei. Nevertheless, 10 per cent of the total nuclear phospholipids remain bound to the chromatin. The comparative analysis of both nuclei and chromatin shows a difference in phospholipids and fatty acid composition. Thus, the chromatin-associated phospholipid cannot be attributed simply to contaminating nuclear membrane. This is supported by the autoradiographic study of semi-thin sections of interphase nuclei from root apices of Vicia faba in which [3H] ethanolamine is clearly localized in the chromatin and nucleolar regions of the nuclei.     

ACTION OF MITOCHONDRIAL DNAASE I IN DESTROYING THE CAPACITY OF ISOLATED CELL NUCLEI TO FORM GELS

1. DNA prepared from non-gelable rat liver nuclei isolated in the presence of disrupted mitochondria at pH 6.0, has been compared with DNA obtained from gelable nuclei isolated at pH 4.0. The DNA of the non-gelable nuclei is partially depolymerized relative to the DNA of the gelable nuclei. 2. It has been found that sufficiently small quantities of crystallized DNAase I can cleave a very large part of the DNA of gelable nuclei isolated at pH 4 from the residual protein of these nuclei without causing extensive depolymerization of the DNA. At the same time the gelable nuclei are rendered non-gelable. 3. Partially purified DNAase II can also render gelable nuclei isolated at pH 4 non-gelable, and in so doing presumably also cleaves the DNA from the residual protein of the nuclei. 4. Mitochondrial DNAase I appears to be the enzyme responsible to a large extent for the cleavage of DNA from the residual protein of gelable rat liver cell nuclei with concomitant destruction of the gel-forming capability of these nuclei, when the nuclei are subjected to the action of disrupted mitochondria at pH 6.0 during the isolation procedure. 5. Mitochondrial DNAase II does not appear to exert appreciable action on nuclei during the course of isolation of the nuclei at pH 6.0 in the presence of disrupted mitochondria. 6. It is probable that DNAase I is not the sole enzyme responsible for destroying the gelability of nuclei isolated at pH 6.0 in the presence of disrupted mitochondria. Protease may be involved. 7. Sodium dodecyl sulfate at pH 6.0–6.3 cleaves the DNA of isolated gelable nuclei from the residual protein of these nuclei over a period of 2 to 3 hours. At pH 7.0–7.5, however, there is negligible cleavage over a period of 96 hours. 8. If non-gelable nuclei are isolated at pH 6.0 in the presence of disrupted mitochondria, DNA subsequently can be removed from them by the use of detergent at pH values ranging from 6.0–7.5 without the necessity of incubation in the detergent solution, since the DNA had already been detached from the residual protein by the action of the mitochondrial enzyme system during isolation of the nuclei.     

In vitro synthesis and stability of RNA in isolated nuclei from bovine lymphocytes

Nuclei from Concanavalin A-stimulated lymphocytes (30 hr after Con A addition) incorporate up to 5 times more (3-H)UTP into RNA than nuclei from resting lymphocytes. The incorporation kinetics is linear for almost 60 min. 14–20% of the $\text{in vitro}$ labeled RNA is polyadenylated. Poly(A) (?)RNA from both types of nuclei sediments from 4–5S up to more than 30S on sucrose gradients. Nuclei from stimulated cells synthesize about double the amount of RNA larger than 18S than nuclei from resting cells. The same holds for poly(A) (+)RNA. Poly(A) (?) RNA labeled during 10 min in both types of nuclei is stable during a 30 min chase. Under the same conditions poly(A) (+)RNA in nuclei from resting cells is degraded to about 50% during the chase whereas it is stable in nuclei from stimulated cells.     

Reactivation of DNA Replication in Nuclei from Terminally Differentiated Cells: Nuclear Membrane Permeabilization Is Required for Initiation inXenopusEgg Extract

We have usedXenopusegg extract to investigate the requirements for reactivation of DNA replication in nuclei isolated from terminally differentiated chicken erythrocytes. Previous work has shown that reactivation of erythrocyte nuclei in egg extract is accompanied by chromatin decondensation, nuclear envelope reformation, and the accumulation of egg lamin, L_III. However, in those studies, erythrocyte nuclei were prepared by methods that were not designed to maintain the selective permeability of the nuclear membrane, and as such, it is not clear if loss of nuclear membrane integrity played a role in the reactivation process. Therefore, the purpose of this study was to determine if changes in nuclear membrane permeability are required for reactivation of erythrocyte nuclei in egg extract. Nuclei with intact nuclear membranes were prepared from erythrocytes with streptolysin O and permeable nuclei by treatment of intact nuclei with the detergent Nonidet-P40. Like permeable nuclei, most intact nuclei decondensed, imported nuclear protein, and accumulated lamin L_IIIfrom the extract. However, unlike permeable nuclei, which replicated extensively in the extract, few intact nuclei initiated replication under the same conditions. These data demonstrate that permeabilization of the nuclear membrane is required for reactivation of DNA replication in terminally differentiated erythrocyte nuclei by egg extract and suggest that loss of nuclear membrane integrity may be a general requirement for replication of quiescent cell nuclei by this system.     

The isolation of nuclei from tissue-cultured plant cells

A method for the isolation of nuclei from tissue-cultured plant cells is presented. The method employs polyamines as stabilizing agent and uses Percoll for the purification of the nuclei in density gradients. It is applicable to large amounts of tissue and is reasonably quick (500 g of tissue can be processed within 2–3 h). The purified nuclei have retained their morphological characteristics as demonstrated by phase contrast, as well as electron micrographs. High molecular weight DNA can be isolated from these nuclei and histones extracted from the purified nuclei display the expected gel electrophoretic pattern. The retainment of the nucleosomal arrangement is demonstrated by digestion with micrococcal nuclease and subsequent analysis of the DNA fragments on agarose gels. The purified nuclei contain high activities of several nucleus-specific enzymes such as α-amanitin-sensitive and insensitive RNA polymerases, protein kinases, poly-ADP-ribosylating enzymes and DNA polymerases.     

Isolation of skeletal muscle nuclei

A method for isolation of nuclei from rat skeletal muscle is described. The tissue is homogenized in the presence of Triton X-100, resulting in release of the nuclei from the muscle fibers. A crude nuclear pellet is prepared by differential centrifugation, and further purification is accomplished by centrifugation through dense sucrose. Bovine serum albumin is used to stabilize the nuclei during the isolation procedure. The isolated nuclei have been characterized microscopically and by chemical and enzymatic assay procedures.     

RNA synthesis in nuclei isolated from early embryos of Xenopus laevis.

1. Rates of RNA synthesis in isolated Xenopus embryo nuclei decrease from blastula through gastrula and neurula stages to hatching tadpoles. 2. In blastula and gastrula nuclei, net synthesis of RNA continues for over 30 min, both in the presence of KCl at 0.4 M and in its absence. In nuclei from later stages, net synthesis continues for only about 10 min in the absence of KCl. 3. At low ionic strength, RNA synthesis in all nuclei is greater with optimum Mg-2+ (6 mM) than with optimum Mn-2+ (1 mM). At high ionic strength the reverse is true. 4. An unusual feature, which gradually disappears as development proceeds, is that curves relating RNA synthesis to KCl concentration show a peak at 0.1 M KCl. In blastula nuclei, RNA synthesis is more rapid at 0.1 M KCl than at 0.4 M. 5. This peak at low ionic strength is not observed in the presence of the initiation inhibitor rifamycin AF/013. It is concluded that the peak arises from initiation of RNA synthesis by an excess of RNA polymerases bound non-specifically to the isolated nuclei. The residual synthesis, representing elongation of chains that were initiated in vivo, still declines as development progresses. 6. In blastula nuclei, over half of the RNA synthesis is effected by polymerase II (inhibited by alpha-amanitin), the proportion remaining roughly constant with increasing ionic strength. In neurula nuclei, the proportion rises from about one-half to three-quarters. The initiation-dependent peak in blastula and gastrula nuclei is contributed by both alpha-amanitin-sensitive and alpha-amanitin-resistant enzymes.     

Studies on ribonucleic acid and homopolyribonucleotide formation in neuronal, glial and liver nuclei

1. Various types of nuclear preparations, with different ratios of neuronal to glial nuclei, were isolated from guinea-pig cerebral grey matter and ox cerebral grey matter and white matter. Conditions appropriate for the separate assay of RNA and poly A formation were described. Comparative rates of RNA and poly A formation were studied in cerebral and liver nuclei. 2. RNA polymerase activity per nucleus is higher in neuronal nuclei than in glial nuclei. In liver nuclei, the activity is much lower than in cerebral nuclei. The physical relationship between RNA polymerase and deoxyribonucleoprotein seems to differ in neuronal, glial and liver nuclei. 3. Poly A polymerase activity in liver nuclei is selectively activated by Mn(2+) and inhibited by GTP, CTP and UTP. On a DNA basis, the activity in an aggregate enzyme is the same as in intact nuclei. Poly A polymerase activity per nucleus is much higher in liver nuclei than in neuronal nuclei. Glial nuclei show an intermediate activity. 4. It is suggested that, in neuronal nuclei, the synthesis of RNA is more prominent than that of poly A under conditions where both polymers are formed simultaneously. This contrasts with liver nuclei, where more poly A is made than RNA. 5. In neuronal nuclei, the rate of CTP incorporation is much higher than in glial and liver nuclei. This incorporation is most probably due to poly C synthesis.     

A rapid procedure for isolating hemopoietic cell nuclei

A new method for isolating cell nuclei is described which involves freezing and thawing cells in 2% Tween 40, then gentle homogenization to release nuclei, followed by immediate microcentrifugation through 50% sucrose. Purified nuclei were obtained in 3 min and yields of 78-95% were obtained from a variety of human hemopoietic cells. Electron microscope analysis of nuclei obtained from HL60 cells showed that 89% of the nuclei were intact and have an appropriate morphology. A low level of contamination with other organelles was revealed by electron microscopy and by using specific assays for plasma membrane, mitochondria, lysosomes, Golgi membrane, and endoplasmic reticulum (0.5-5.5%). The value of the technique is that nuclear proteins and small metabolites which might be lost by rapid leakage from isolated nuclei and the possibility of biochemical modification of cellular constituents are minimized by using a rapid isolation procedure.     

Replication of Nuclei from Cycling and Quiescent Mammalian Cells in 6-DMAP-TreatedXenopusEgg Extract

Nuclear membrane permeabilization is required for replication of quiescent (G0) cell nuclei inXenopusegg extract. We now demonstrate that establishment of replication competence in G0 nuclei is dependent upon a positive activity present in the soluble egg extract. Our hypothesis is that G0 nuclei lose the license to replicate following growth arrest and that this positive activity is required for relicensing DNA for replication. To determine if G0 nuclei contain licensed DNA, we used the protein kinase inhibitor, 6-dimethylaminopurine (6-DMAP), to prepare egg extracts that are devoid of licensing activity. Intact nuclei, isolated from mammalian cells synchronized in G1-phase (licensed), G2-phase (unlicensed), and G0 were permeabilized and assayed for replication in 6-DMAP-treated and untreated extracts supplemented with [α-³²P]dATP or biotinylated-dUTP. Very little radioactivity was incorporated into nascent DNA in each nuclear population; however, nearly all nuclei in each population incorporated biotin in 6-DMAP extract. The pattern of biotin incorporation within these nuclei was strikingly similar to the punctate pattern observed within nuclei incubated in aphidicolin-treated extract, suggesting that initiation events occur within most replication factories in 6-DMAP extract. However, density substitution and alkaline gel analyses indicate that the incorporated biotin within these nuclei arises from a small number of active origins which escape 6-DMAP inhibition. We conclude that 6-DMAP-treated egg extract cannot differentiate licensed from unlicensed mammalian somatic cell nuclei and, therefore, cannot be used to determine the “licensed state” of G0 nuclei using the assays described here.     

Small angle scattering of cell nuclei

Neutron and X-ray small angle scattering techniques have been applied to study chromatin structure inside different types of cell nuclei. Scattering from genetically inactive chicken erythrocyte nuclei exhibits a maximum at Q = 0.1-0.15 nm-1 which cannot be observed by studying isolated chromatin derived from the same kind of cells. In highly active transcribing rat liver nuclei such a nuclear pattern is absent. The radius of gyration of isolated "superbeads" was determined. It is discussed whether the characteristic maximum of the nuclei originates from this superstructural organisation of chromatin. Rat liver nuclei were fractionated on sucrose gradients in order to determine whether the absence of the extra maximum in scattering profiles of these nuclei is due to overlapping effects of different chromatin organisation in the various cell types of the liver. As compared to unfractionated nuclei no strong deviations in the scattering profiles of the fractions could be observed. Erythrocyte nuclei were dialysed in buffers differing in the ionic strength of monovalent cations. The typical maximum from the nuclei is shifted from 60 nm (very low salt concentration) to about 35 nm (physiological ionic strength) and is linearly proportional to the decreasing radius of the nuclei. In conclusion, chromatin structure inside the nucleus has a scattering maximum due to an ordered packing of the fibres which is absent in nuclei with high genetic activity.     

Karyometric marker features in fine needle aspirates of follicular adenoma of the thyroid

Karyometric measurements were performed on fine needle aspirates of clearly identifiable adenomatous areas and adjacent normal-appearing areas in the surgical specimens from ten patients with follicular adenomas of the thyroid. Similar measurements were made on aspirates from nine patients free of thyroid disease. A total of 95 karyometric features were evaluated for each nucleus. Analysis of variance of optical density values did not show a significant difference between the three types of nuclei. Discriminant analysis selected seven karyometric features that produced a statistically highly significant separation of adenoma nuclei from control nuclei. A similar analysis selected six features that produced a statistically highly significant discrimination of normal-appearing nuclei from control nuclei. The validity of these markers for distinguishing control nuclei from adenoma nuclei and normal-appearing nuclei adjacent to adenomas was demonstrated by analysis in further training and test sets. These findings parallel those previously demonstrated for invasive and microinvasive follicular carcinomas of the thyroid.     

Automatic segmentation of cell nuclei in Feulgen-stained histological sections of prostate cancer and quantitative evaluation of segmentation results

Digital image analysis of cell nuclei is useful to obtain quantitative information for the diagnosis and prognosis of cancer. However, the lack of a reliable automatic nuclear segmentation is a limiting factor for high-throughput nuclear image analysis. We have developed a method for automatic segmentation of nuclei in Feulgen-stained histological sections of prostate cancer. A local adaptive thresholding with an object perimeter gradient verification step detected the nuclei and was combined with an active contour model that featured an optimized initialization and worked within a restricted region to improve convergence of the segmentation of each nucleus. The method was tested on 30 randomly selected image frames from three cases, comparing the results from the automatic algorithm to a manual delineation of 924 nuclei. The automatic method segmented a few more nuclei compared to the manual method, and about 73% of the manually segmented nuclei were also segmented by the automatic method. For each nucleus segmented both manually and automatically, the accuracy (i.e., agreement with manual delineation) was estimated. The mean segmentation sensitivity/specificity were 95%/96%. The results from the automatic method were not significantly different from the ground truth provided by manual segmentation. This opens the possibility for large-scale nuclear analysis based on automatic segmentation of nuclei in Feulgen-stained histological sections.     

Control of rRNA transcription in liver of mice exposed to nutritional changes: initiation frequency may be a regulatory mechanism

Shortly after feeding protein-depleted mice on a meal containing proteins, the RNA polymerase I activity in isolated liver nuclei shows a two fold to threefold activation over the basal value in nuclei of either normal or protein-depleted mice. This activation can be accounted for by the increase in the number of growing rRNA chains. Moreover, the template-bound RNA polymerase I fraction in nuclei from re-fed mice is about three times that from protein-depleted animals. An excess of template- unbound enzyme was found in liver nuclei from animals under either nutritional condition. Shortly after inhibition of protein synthesis by pactamycin administration to re-fed mice, the number of transcribing RNA polymerase I molecules in liver nuclei decreases to the basal level found in nuclei from protein-depleted mice, while in the latter, protein synthesis inhibition has no effect. These results support the suggestion that short-lived proteins may enhance the initiation frequency by RNA polymerase I after re-feeding.     

Is there "Metabolic" DNA in the Mouse Seminal Vesicle?

This study was designed to answer the question: Is H³-thymidine uptake by nuclei of the mouse seminal vesicle evidence for DNA synthesis and mitosis, or does it signify some "metabolic" function of DNA unrelated to chromosome duplication? Mice were given an intraperitoneal injection of H³-thymidine. Six hours later Feulgen squashes of the seminal vesicle epithelium were made and covered with autoradiographic stripping film. The silver grains above labeled nuclei were counted, and the Feulgen dye contents of these same nuclei were determined photometrically after removal of the grains from the emulsion. Unlabeled nuclei were also measured. The dye contents of non-radioactive nuclei form a unimodal distribution, indicating that polyploidy is absent from this tissue. The radioactive nuclei fall into two groups. In the first, the average dye content is the same as that of the cold nuclei (2C). In the second, the values range from 2C to 4C. In the 2C to 4C group the grain count is proportional to the dye content, showing that incorporation is correlated with synthesis. The radioactive 2C nuclei arose by mitosis during the course of the experiment. This is shown by the following facts: (1) They frequently occur in pairs. (2) They average smaller than unlabeled 2C nuclei. (3) Their average grain count is approximately half that of the 4C nuclei. (4) Labeled division figures are found. (5) A mitotic rate estimated from the number of labeled 2C nuclei accords reasonably well with one based on the number of observed mitoses. Since the incorporation of thymidine accompanies DNA synthesis and precedes mitosis, there is no reason to postulate a special "metabolic" DNA in this tissue.     

The INTACT method for cell type-specific gene expression and chromatin profiling in Arabidopsis thaliana

Genomic studies of cell differentiation and function within a whole organism depend on the ability to isolate specific cell types from a tissue, but this is technically difficult. We developed a method called INTACT (isolation of nuclei tagged in specific cell types) that allows affinity-based isolation of nuclei from individual cell types of a tissue, thereby circumventing the problems associated with mechanical purification techniques. In this method nuclei are affinity-labeled through transgenic expression of a biotinylated nuclear envelope protein in the cell type of interest. Total nuclei are isolated from transgenic plants and biotin-labeled nuclei are then purified using streptavidin-coated magnetic beads, without the need for specialized equipment. INTACT gives high yield and purity of nuclei from the desired cell types, which can be used for genome-wide analysis of gene expression and chromatin features. The entire procedure, from nuclei purification through cDNA preparation or chromatin immunoprecipitation (ChIP), can be completed within 2 d. The protocol we present assumes that transgenic lines are already available, and includes procedural details for amplification of cDNA or ChIP DNA prior to microarray or deep sequencing analysis.