Assembly of transcriptionally inactive chromatin in vitro.

We have successfully uncoupled the previously interlocked activities of chromatin assembly and in vitro transcription promoted by the Xenopus oocyte S-150 cell-free extract. Our isolated fraction catalyzes extensive chromatin assembly measured both by changes in DNA topology and Micrococcal nuclease digestions. The assembly of chromatin is slowed by the exogenous addition of ATP. In the absence of exogenously added ATP, the fraction forms a chromatin template that is transcriptionally inert. Addition of small amounts of the HeLa cell extract (S-100) converts these templates into transcriptionally active ones without disrupting the chromatin structure. Our protocol defines a method for the isolation of a fraction from the Xenopus cell free extract that catalyzes the assembly of transcriptionally inactive chromatin. We characterize this reaction and establish conditions for the transcriptional activation of these inactive minichromosomes.

Influence of histone acetylation on the solubility, H1 content and DNase I sensitivity of newly assembled chromatin.

In a previous report [Annunziato, A.T. and Seale, R.L. (1983) J. Biol. Chem. 258:12675] a novel intermediate in chromatin assembly was described (detected by labeling new DNA in the presence of the deacetylase inhibitor sodium butyrate), which retained approximately 50% of the heightened sensitivity of newly replicated chromatin to DNaseI. It is now reported that nucleosomes replicated in butyrate are considerably more soluble in the presence of magnesium, relative to chromatin replicated under control conditions, and that this heightened magnesium-solubility is reflected in a concomitant increase in the preferential solubility of nucleosomes containing newly synthesized core histones. This differential solubility was accompanied by a 5- to 6-fold depletion of histone H1, and was completely abolished by the selective removal of H1 from isolated nuclei. The removal of H1 also markedly reduced the preferential DNaseI sensitivity of chromatin replicated in butyrate. Further, when mononucleosomes of control and (acetylated) nascent chromatin were compared, no differences in DNaseI sensitivity were detected. These results provide evidence that the interactions between newly assembled nucleosomes and histone H1 are altered when histone deacetylation is inhibited during chromatin replication, and suggest a mechanism for the control of H1 deposition during nucleosome assembly in vivo.

[Use of very low density light fluorescence microscopy: study of real-time of the development of chromatin of unicellular mouse embryos]. / Utilisation de la microscopie de fluorescence à très bas niveau de lumière: étude en temps réel de l'évolution de la chromatine d'embryons unicellulaires de souris.

Video enhanced fluorescence microscopy coupled to digitized image processing was used to follow the dynamics of chromatin changes in live one cell mouse embryos. The experimentation on live material was made possible through the use of very low concentrations of the DNA specific fluorophore, Hoechst 33342, and very low irradiation intensities, which maintain a good viability of embryos.

Step-wise progression of mammalian 10-kb DNA replication intermediates to mature chromatin.

In mammalian DNA synthesis the primary replication intermediates are joined to larger intermediates. After the joining process is complete one can detect a distinct stage called the post-elongation stage. Furthermore a 10-kb DNA1 population is detected before the post-elongation stage whereas a 10-kb DNA2 population is part of this stage DNA. When cells are treated with 3-aminobenzamide, an inhibitor of poly(ADP-ribose) synthetase, an altered post-elongation-stage DNA was detected, which does not contain 10-kb fragments. The step(s) affected by 3-aminobenzamide prevents the appearance of 10-kb DNA in the post-elongation stage. The drug effect is reversible with the appearance of 10-kb DNA in the post-elongation stage when the cells are washed free of drug. Hence there is a step-wise progression from 10-kb DNA, via the post-elongation stage, to mature chromatin.

Exclusion chromatography with controlled-pore glass beads to isolate Chlorella chromatin and its applications.

A simple and rapid method was developed to isolate chromatin from the unicellular alga, Chlorella, by exclusion chromatography utilizing controlled-pore glass beads. This method takes advantage of the giant size of the chromatin supramolecules and does not require the preliminary isolation of cell nuclei. In order to raise the histone yield, commercially available materials were silanized with dimethyldichlorosilane. The isolated algal chromatin had properties similar to those of other organisms, and the histones contained all five components found in calf thymus. A hierarchy of the higher order structures was also observed in the algal chromatin. This method can be used for the study of chromatin in various cell types, especially in microbial cells, from the viewpoints of not only mere preparation but also cell dynamics and fractionation in relation to the specific components or activities. Some application examples are presented.

Interrelationships of protein and DNA syntheses during replication of mammalian cells.

During the replication of chromatin, the syntheses of the histone protein and DNA components are closely coordinated but not totally linked. The interrelationships of total protein synthesis, histone protein synthesis, DNA synthesis, and mRNA levels have been investigated in Chinese hamster ovary cells subjected to several different types of inhibitors in several different temporal combinations. The results from these studies and results reported elsewhere can be brought together into a consistent framework which combines the idea of autoregulation of histone biosynthesis as originally proposed by W. B. Butler and G. C. Mueller (Biochim. Biophys. Acta 294:481-496, 1973] with the presence of basal histone synthesis and the effects of protein synthesis on DNA synthesis. The proposed framework obviates the difficulties of Butler and Mueller's model and may have wider application in understanding the control of cell growth.

[Effect of ethanol on the synthesis of chromatin proteins of the nuclei of rat liver cells]. / Vliianie étanola na sintez khromatinovykh belkov iader kletok pecheni krys.

The synthesis of nuclear chromatin proteins of rat liver cells was studied after a momentary and chronic administration of ethanol (3 g/kg). The 14C-acetaldehyde penetration to the nuclei and its binding to chromatin protein components were also studied. The momentary administration of ethanol inhibits the synthesis of histons and reenhances the incorporation of radioactive precursors into DNA labile-bound nonhiston proteins. The reduced level of the synthesis of all the studied chromatin proteins was observed after the chronic administration of ethanol.

In vitro rabbit mammary gland chromatin replication studied by micrococcal nuclease digestion.

Isolated nuclei from pregnant rabbit mammary glands were labelled with [3H]dTTP under conditions for DNA synthesis and subsequently digested with micrococcal nuclease. Replicating chromatin was found to exhibit increased susceptibility towards the nuclease. Analysis of chromatin digestion products by sucrose density gradient centrifugation demonstrated the association of in vitro replicated DNA with nucleosomes. Furthermore, the distribution of DNA polymerizing activity was studied in isolated nuclease-digested mammary gland chromatin. About 90% of all recovered nuclear DNA polymerizing activity cosedimented with nucleosomal particles, mainly with mononucleosomes. The distribution of DNA polymerases alpha and beta in chromatin isolated from the mammary glands of pregnant and lactating rabbits was compared. In these physiological states the mononucleosome-associated DNA polymerase alpha activity varied in accordance with the rate of DNA synthesis.

Analysis of the chromatin assembled in germinal vesicles of Xenopus oocytes.

We have injected circular DNA, labeled with 32P at a single restriction site, into germinal vesicles of Xenopus laevis oocytes in order to study the nucleosome arrangement on the assembled minichromosomes. Two types of genes were used in these studies, the somatic 5 S RNA gene unit of Xenopus borealis and the histone gene unit of Drosophila melanogaster. We find that injections of labeled DNA alone, at 1 ng DNA per oocyte, results in irregularly spaced nucleosomes and partially supercoiled DNA molecules. However, perfectly spaced nucleosomes are assembled and fully supercoiled DNA is recovered if 5 to 20 nanograms of cold vector DNA is coinjected with the labeled DNA. At the optimum chromatin assembly conditions, the nucleosomes are perfectly spaced with a 180 base-pair periodicity, but they are randomly positioned on the DNA. The assembly of a periodic chromatin structure is accompanied by a dramatic enhancement in the expression of the injected 5 S RNA gene.

Correlation of patterns of nuclear protein synthesis with differentiation of mitogen-stimulated B-lymphocytes.

Nuclear proteins synthesised by mouse lymphocytes stimulated by the B-lymphocyte mitogen lipopolysaccharide have been analysed by 2-dimensional polyacrylamide gel electrophoresis. It has been shown that the rate of synthesis of both nucleoplasmic proteins and nonhistone chromatin proteins is stimulated dramatically by LPS and many nuclear proteins are synthesised that were undetectable in resting lymphocytes. The majority of these proteins are synthesised by lymphoblasts and plasma cells, the lymphocytes that remain small after LPS stimulation synthesizing relatively few proteins. Two chromatin proteins that have previously been shown to appear in the nucleus within 4h of mitogen stimulation exhibit a continuous increase in their rate of synthesis during lymphocyte differentiation. The possible roles of these proteins in differentiating lymphocytes are discussed.

Effects of chromatin protein fractions on transcriptional activity of chicken thrombocyte and erythrocyte chromatin.

1. Chromatin proteins of chicken thrombocytes and erythrocytes were separated into three fractions by successive extraction with 5 M urea containing various salt concentrations and pH values. Molecular composition of protein fractions was determined by SDS-polyacrylamide gel electrophoresis. 2. The efficiences of the chromatin residues after sequential protein extractions as well as those of reconstituted DNA-protein fraction complexes, in serving as a template for the in vitro RNA synthesis were measured in order to identify the effect of each fraction. 3. The different involvement of chromatin protein fractions of template properties of thrombocyte and erythrocyte chromatin was stated.

[Histone competition for DNA and its possible role in the self-assembly of eu- and heterochromatin]. / Konkurentsiia gistonov za DNK i ee vozmozhnaia rol' v samosborke éu- i geterokhromatina.

Electrophoretic analysis of histones bound to DNA and remaining free in the mixtures of DNA with the total histone of chromatin in a medium of physiological ionic strength has shown that even the minimal weight excess of the total histone with reference to DNA (1.1:1) leads to the formation of nucleohistone impoverished in HI fraction because of histone competition for DNA. Within the histone/DNA ratio equal to 1.4, H3, H4, H2A and H2B are bound to DNA without competition, i.e. at a ratio in which they are added to DNA. Provided the histone/DNA ratio is higher in the mixture, there form nucleohistones enriched with H3 and H4 fractions. The role of histone competition for DNA in eu- and heterochromatin assembly is discussed.

Some properties of chromatin synthesized by mouse-L-cells temperature-sensitive in DNA replication.

When ts A1S9 mouse L-cells are incubated at the nonpermissive temperature (38.5 degrees) DNA synthesis proceeds at the normal rate for 6 to 8 h; it then declines to attain 1 to 5% of this rate after 24 h. General protein synthesis from precursor leucine is relatively unaffected by the high temperature. In contrast, protein formation from lysine (and arginine) remains unchanged for 12 to 15 h after temperature upshift. It then drops and plateaus at about 25% of the initial rate after 32 h. The chromatin protein and DNA are fully conserved in ts A1S9 cells incubated at 38.5 degrees for at least 24 h after full expression of the ts defect. Temperature inactivation of the ts A1S9 gene product results in inhibition of de novo formation of chromatin. This is evidenced by coordinate suppression of incorporation of dThd and of lysine and arginine into chromatin-bound DNA and histone, respectively.

Serum stimulation of human fibroblasts: effect on non-histones of nuclear ribonucleoprotein and chromatin.

In quiescent human fibroblasts stimulated to proliferate by fresh medium plus 15% serum, no changes were seen in the incorporation of 3H tryptophan into the protein of nuclear ribonucleoprotein during the first three hours following re-feeding. This was in contrast to non-histone chromosomal proteins where the incorporation increased by 90% within ten minutes. The density of the formaldehyde fixed nuclear ribonucleoprotein in CsCl was 1.43-1.44 g/ml and this also did not change following stimulation. The electrophoretic profile of the proteins of nuclear ribonucleoprotein on SDS gels exhibited a predominant band corresponding to a molecular weight of 44,000 closely trailed by a band at 47,000 and other bands at higher molecular weight. This pattern was not altered by serum stimulation and the same was true for the more complex electrophoretic profile of the chromatin proteins. Following a 10-minute pulse of 3H-tryptophan at ten minutes after stimulation, there was a selective increase in the labeling of non-histone chromosomal protein of molecular weight 59,000; no change was seen in the labeling of any protein of nuclear ribonucleoprotein.