Bead-shift isolation of protein--DNA complexes on a 5S RNA gene.

Specific protein-DNA complexes formed on a Xenopus 5S RNA gene were isolated and characterized using a novel technique. A DNA template reversibly immobilized on paramagnetic beads was used to capture, affinity purify, and concentrate protein--DNA complexes formed in a whole cell extract. The complexes were then released from the beads in a soluble and transcriptionally active form via restriction enzyme digestion of the DNA. A band-shift gel was used to separate and obtain the DNase I footprints of five individual complexes. Three of the complexes resulted from the independent binding of two proteins, TFIIIA and an unidentified protein binding to a large region just downstream of the 3' end of the gene. Two more slowly migrating complexes contained an additional large central protected region covering most of the gene. The most slowly migrating complex displayed protein interactions over the 5' flanking sequences. The formation of two of these complexes was shown to be dependent on TFIIIC activity. The correlation between transcriptional activity and the formation of these complexes suggests that the observed protein--DNA interactions are important for transcription of 5S RNA genes.

Small, heat-stable, DNA-binding proteins from Caulobacter crescentus.

From a heterogenous cell population of Caulobacter crescentus a deoxyribonucleoprotein fraction (DNP) was obtained by the modified technique of Sjåstad et al. (1982). Under the electron microscope DNP had a smooth fibrillar structure. The chemical properties of the proteins associated with the DNA were similar to those of other bacteria. An abundant, heat-stable, basic and DNA-binding protein, termed HCc, which has a molecular weight of 13.4 kD may be an analogue of the HU-histone-like protein from Escherichia coli.

Differential decondensation of mitotic chromosomes during hypotonic treatment of living cells as a possible cause of G-banding: an ultrastructural study.

The chromosomal ultrastructure of Chinese hamster cells treated with 0.075 M KCl - a solution ordinarily used for making preparations of spread chromosomes - was studied. The hypotonic treatment was shown to result in differential decondensation of chromosomes which consists in the uneven distribution of deoxyribonucleoprotein (DNP) fibrils along chromatids. Fixation of cells with methanol acetic acid causes an abrupt restructuring of chromosomes. However, the DNP preserves its uneven distribution along chromatids. As seen on ultra-thin sections of marker nucleolus organizer chromosomes, the densely packed regions may correspond to G-bands detected in the selfsame chromosomes by standard methods of differential staining. The results suggest that the capacity of chromosomes for differential staining is based on the different resistance of G- and R-bands to the decondensing action of hypotonic solutions on living cells.

[Local homology of amino acid sequences of histones H3, H4 and the protein repressor lambda Cro. Possible conformation of homologous histone sites in DNP]. / Lokal'naia gomologiia aminokislotnykh posledovatel'nostei gistonov H3, H4 i belka repressora lambda Cro. Vozmozhnaia konformatsiia gomologichnykh uchastkov gistonov v DNP.

A mathematical analysis of amino acid sequences was carried out with a view of detecting possible homology between histones H3 and H4 and repressor-activator proteins of prokaryotes according to the A. I. criterion which reflects the similarity of their primary structure. It was found that the sites of eukaryotic histones H3 (102-123) and H4 (68-85) and site alpha 3 (24-25) of the prokaryotic repressor protein lambda Cro, i. e., the site of protein interaction with DNA, reveal a statistically significant homology. The A. I. value for the H3 site of lambda Cro is 3.37, that for the H4 site of calf thymus and sea horse is 3.28. The amino acid sequences of these proteins in the alpha 2-alpha 3 site, i. e., the site in which the homology between amino acid sequences of histones and DNA-binding proteins had been established previously, with regard to similarity of their secondary structure of the helix-turn-helix type, were analyzed. A pairwise comparison of H3 and protein lambda Cro showed that the A. I. value for histones H3 from various sources is approximately 2.7; however, the homology of the alpha 2 site is lower than that of site alpha 3. It is concluded that there exists an evolutionary relationship between homologous segments of histones H3 and H4 and protein lambda Cro, which can be preserved in order to maintain a definite secondary structure, presumably for binding to DNA.

[Structural changes of the chromatin DNA in situ in partial deproteinization of DNP by a 0.6 M NaCl solution. A polarization-fluorescence microscopic study]. / Strukturnye izmeneniia DNK khromatina in situ pri chastichnoi deproteinizatsii DNP 0.6 M rastvotom NaCl. Poliarizatsionno-fluorestsentnoe mikroskopicheskoe issledovanie.

By means of polarization fluorescence microscopy changes in polarization degree of fluorescence of DNA-bound of Acridine orange in situ were observed. A marked decrease in polarization degree of chromatin fluorescence was found after the treatment of cell nuclei with 0.6 M NaCl. It shows that the removal of histone H1 from the chromatin results in destabilization of its structure. The data obtained show that the measurements of polarization degree of fluorescence allow to obtain relevant information about structural changes in DNA chromatin.

[Localization of lysine residue in histone H3 forming the thymine-lysine cross-link after UV-irradiation of deoxyribonucleoprotein]. / Lokalizatsiia ostatka lizina v gistone H3, obrazuiushchego timin-lizinovuiu sshivku pri obluchenii dezoksiribonukleoproteida UF-svetom.

A thymine-modified derivative of histone H3 formed as a result of thermal treatment of UV-irradiated (lambda = 254 nm) solution of deoxyribonucleoprotein from calf thymus at low ionic strength was isolated. The peptides obtained by tryptic hydrolysis of modified histone H3 were separated by high pressure liquid chromatography. The amino acid sequence of the peptide containing a lysine residue with covalently linked thymine was determined by the Edman method. It was found that Lys localized at the N-terminus of the histone H3 molecule interacts with DNA within the composition of the deoxyribonucleoprotein.

[Principle of the supramolecular stacking of the cellular deoxyribonucleoprotein complex]. / O printsipe nadmolekulianoi ukladki dezoksiribonukleoproteidnogo kompleksa kletki.

Using cytofluorimetry with acridine orange staining and a modified thermal denaturation technique of cellular DNP, it has been shown that chromatin melting profiles of normal human nuclei (from lymphocytes and granulocytes) have distinct regularities. It is believed that these regularities reflect specific supramolecular chromatin organization. Parallel comparative analysis performed using electrophoretic fractionation and isoelectric focussing of nuclear proteins has revealed that: 1) peculiarities of chromatin melting profiles are independent of the quantity and molecular weights of chromatin proteins; 2) the lack of principal differences in chromatin melting profiles and the data on isoelectric points of nuclear proteins of granulocytes and lymphocytes from the same patient indicate that specific supramolecular organization of DNP-complex depends on the chromatin protein charge.

A comparative study on proteins released from metaphase chromosomes after digestion with restriction endonucleases and deoxyribonuclease I.

Extensive digestion of Chinese hamster metaphase chromosomes with Alu I, Hae III and Hinf I released up to 40 distinct chromosomal proteins. Some of the proteins released by Hae III or Hinf I were enriched in the protein moiety liberated by Alu I but several proteins released by Hae III were not released by Alu I digestion. The amount of chromosomal protein released by deoxyribonuclease I (DNase I) was comparable to that liberated by the three restriction enzymes so far tested, while only four abundant protein species were detectable in the protein moiety released by DNase I. Two of them with molecular weights of 58,000 and 50,000 were also released by the three restriction enzymes and are similar in size to those found previously in the core-like structure of histone-depleted chromosomes.

[Characteristics of protein-DNA association in chromatin from epidermal cells in psoriasis]. / Kharakteristika assotsiatsii belka i DNK v khromatine kletok épidermisa pri psoriaze.

Two forms of nucleoprotein complex alpha and beta were found in human keratinocytes by means of nucleoprotein celite chromatography. They were not observed any principal differences between distribution of chromatin various forms in impaired and normal skin of the patients with psoriasis as well as in skin of healthy persons. Only slight differences were detected in the ratio of radioactive DNA in alpha and beta forms of chromatin from impaired and normal keratinocytes, while tightly-bound DNA was increased in psoriasis. In psoriasis acceleration of keratinocyte cell cycle but not an increase in proliferating cells pool appear to occur.

Nuclease resistance and the enrichment of native nuclear acceptor sites for the avian oviduct progesterone receptor.

High-affinity nucleoprotein acceptor sites for the avian oviduct progesterone receptor (PR) have been enriched by a combination of nuclease digestion and centrifugation. These enriched binding elements exhibited markedly enhanced PR binding on a per mass DNA basis compared to chromatin (20- to 25-fold) or dehistonized chromatin (4- to 5-fold). Electrophoretic analysis of the nuclease-resistant DNA showed that there is a set of DNA fragments of 100-150 base pairs that are protected from digestion. Excessive digestion resulted in smaller DNA fragments and a loss of PR binding activity. The PR binding was saturable using a crude receptor preparation and displayed a competition with the same receptor preparation that was labeled with nonradioactive progesterone. The enhanced binding was also demonstrable using highly purified receptor preparations that exhibit two classes of binding sites both of which are of high affinity and saturable as assessed by Scatchard analyses. These two high-affinity classes of binding sites are shown to be competed by unlabeled purified PR. The nuclease resistance of these nucleoprotein acceptor sites from chromatin is a property similar to the nuclear matrix binding sites suggesting a relationship between these two classes of nuclear acceptor sites.

A 10S particle released from deoxyribonuclease-sensitive regions of HeLa cell nuclei contains the 86-kilodalton-70-kilodalton protein complex.

Digestion of HeLa cell nuclei with micrococcal nuclease or deoxyribonuclease I (DNase I) released the 86-kilodalton-70-kilodalton (kDa) protein complex in particles sedimenting at approximately 10 S in sucrose density gradients. Immunoaffinity-purified 32P-labeled complexes contained 86- and 70-kDa polypeptides with phosphorylated serine residues and DNA fragments, of which the largest was 110 base pairs long. Digestion of nick-translated nuclei with micrococcal nuclease released 32P-labeled 10S particles that were immunoaffinity-purified; they contained labeled 110-base-pair DNA fragments. The micrococcal nuclease digests were analyzed by two-dimensional electrophoresis, which separated nucleosomes in the first dimension and the associated proteins in the second. Western blots of the separated proteins showed that the 86-kDa-70-kDa complex was associated with the mono-, di-, and trinucleosomes. A more extensive electrophoretic separation revealed that the 10S particle from nick-translated nuclei migrated with a subfraction of the mononucleosomes that lacked H1 histones. These results suggest that the 10S particle which contains the 86-kDa-70-kDa complex is associated with an unfolded nucleosome that is present in DNase I sensitive regions.

Association of the herpes simplex virus regulatory protein ICP4 with specific nucleotide sequences in DNA.

We report that the herpes simplex virus (HSV) transcription regulatory protein designated ICP4 is a component of a stable complex between protein and specific nucleotide sequences in double-stranded DNA formed by addition of exogenous DNA to either a crude extract obtained from HSV-1 infected cells or a partially purified preparation of native ICP4. DNA sites which are bound directly or indirectly to ICP4 have been designated ICP4/protein binding sites. Three independent ICP4/protein binding sites have been identified by DNAse footprinting; two are in the vector pBR322 and one is located approximately 100 nucleotides upstream from the HSV glycoprotein D mRNA cap site. Comparison of the nucleotide sequences in these three sites reveals several regions of homology. We propose that the sequence 5'-ATCGTCNNNNYCGRC-3' (N = any base; Y = pyrimidine; R = purine) forms an essential component of the ICP4/protein binding site.

[Characteristics of sulfhydryl groups of histone H3 from the calf thymus using mercury-containing nitroxyl radicals]. / Kharakteristika sul'fgidril'nykh grupp gistona H3 iz timusa telenka s pomoshch'iu rtut'soderzhashchikh nitroksil'nykh radikalov.

The interaction between total histone and deoxyribonucleoprotein (DNP) preparations from calf thymus with mercury-containing nitroxyl radicals in low ionic strength solutions, 2 M NaCl and urea was investigated. It was found that the label is rapidly incorporated into the SH-groups of histone H3 to produce characteristic EPR signals. Titration of SH-groups within DNP demonstrated that in low ionic strength solutions only one SH-group (presumably, the SH-group of the cysteine residue in position 110) is accessible to the reagents. After dissociation by 2 M NaCl, two SH-groups become titrable; however, the EPR spectra point to differences in the conformational state of these two groups. In 4 M urea, these differences are compensated for by structural disintegration. The spin labels may be used for the analysis of SH-groups under different conditions and at different functional states of nucleoproteins.

Formaldehyde-mediated DNA-protein crosslinking: a probe for in vivo chromatin structures.

Formaldehyde (HCHO) produces DNA-protein crosslinks both in vitro and in vivo. Simian virus 40 (SV40) chromosomes that have been fixed by prolonged incubation with HCHO either in vitro or in vivo (within SV40-infected cells) can be converted to nearly protein-free DNA by limit-digestion with Pronase in the presence of NaDodSO4. The remaining Pronase-resistant DNA-peptide adducts retard the DNA upon gel electrophoresis, allowing resolution of free and crosslink-containing DNA. Though efficiently crosslinking histones to DNA within nucleosomes both in vitro and in vivo, HCHO does not crosslink either purified lac repressor to lac operator-containing DNA or an (A + T)-DNA-binding protein (alpha-protein) to its cognate DNA in vitro. Furthermore, a protein that does not bind to DNA, such as serum albumin, is not crosslinked to DNA by HCHO even at extremely high protein concentrations. These properties of HCHO as a DNA-protein crosslinker are used to probe the distribution of nucleosomes in vivo. We show that there are no HCHO-crosslinkable DNA-protein contacts in a subset of SV40 chromosomes in vivo within a 325-base-pair stretch that spans the "exposed" (nuclease-hypersensitive) region of the SV40 chromosome. This replication origin-proximal region has been found previously to lack nucleosomes in a subset of isolated SV40 chromosomes. We discuss other applications of the HCHO technique, including the possibility of obtaining base-resolution in vivo nucleosome "footprints."

[The role of histones H3 and H1 in the formation of thymine-lysine cross-links during UV-irradiation of deoxyribonucleoprotein]. / Uchastie gistonov H3 i H1 v obrazovanii timin-lizinovoi shivki pri UF-obluchenii deoksiribonukleoproteida.

The effect of UV light (lambda = 254 nm) on calf thymus DNP at low ionic strengths was studied. It was found that at the irradiation doses used the protein in the DNA-protein complex increases as the irradiation dose rises. Thermal treatment and acid hydrolysis resulted in a predominant release of histones H3 and H1 from the complex. Data from liquid high performance chromatography, amino acid analysis, thin-layer chromatography point to the induction by UV-light of a thymine-lysine bond, whose formation involves DNA thymines and histone lysine residues, predominantly H3 and H1 fractions.

Nucleosome reconstruction via phosphorus mapping.

Electron spectroscopic imaging was combined with reconstruction algorithms to derive the three-dimensional structure of the nucleosome core particle to a resolution of 1.5 nanometers. Images of phosphorus distributions within individual nucleosomes were interpreted as projections of a supercoil of DNA. These were used to orient the corresponding individual nucleosome images, making it possible to reconstruct the entire nucleosome in three dimensions. The structure is consistent with known biochemical and biophysical data and explains site-specific nuclease sensitivity, although differing in part with other nucleosome models.

Electron microscopic localization of the chloroplast DNA replicative origins in Chlamydomonas reinhardii.

Chloroplast DNA, isolated from a synchronized culture of Chlamydomonas reinhardii, was digested with restriction endonucleases and examined in the electron microscope. Restriction fragments containing displacement loops (D-loop) were photographed and measured to determine the position of replicated sequences in relation to the restriction enzyme sites. D-loops were located at two positions on the physical map of chloroplast DNA. One replication origin was mapped at about 10 kb upstream of the 5' end of a 16s rRNA gene. The second origin was spaced 6. 5kb apart from the first origin and was about 16.5 kb upstream of the same 16s rRNA. Initiations at those two sites were not always synchronized. Replication initiated with the formation of a D-loop resulting from the synthesis of one daughter strand. After a short initial lag phase, corresponding to the synthesis of 350 +/- 130 bp of one daughter strand, DNA synthesis then proceeded in both directions. Both D-loop regions were preferred binding sites of undetermined protein complexes.

Characterization of the DNA-protein complex at the termini of the bacteriophage PRD1 genome.

DNA of bacteriophage PRD1 has protein P8 at its termini. Extracts of infected cells are able to derivatize P8 in vitro with labeled dGTP. Two early proteins, P1 and P8, products of genes I and VIII, respectively, are the only phage proteins necessary for the formation of the protein P8-dGMP complex. This was shown by complementation of extracts from cells infected with mutants and by use of extracts from cells carrying cloned genes I and VIII. With Escherichia coli mutants that are temperature sensitive for DNA synthesis, it was possible to show that the formation of the protein P8-dGMP complex was dependent upon the host replication apparatus. The analysis of the purified protein P8-dGMP complex by hydrolysis and enzymatic digestion showed that there is a covalent phosphodiester bond between tyrosine and 5'-dGMP.

[DNA-protein complexes of the nuclear matrix of Ehrlich ascites carcinoma cells]. / DNK-belkovye kompleksy iadernogo matriksa kletok astsitnoi kartsinomy Erlikha.

A DNA-protein complex resistant to 8 M urea and 0.1% SDS was obtained by chromatography of nuclear matrix lysate from Ehrlich ascite carcinoma cells on Sepharose 2BCL. Separation of the complex under more severe conditions (4 M guanidine hydrochloride, 5 M urea) on hydroxylapatite resulted in protein and DNA fractions, as well as in two fractions of the DNA-protein complex. One of the fractions of this complex was enriched with single-stranded DNA and contained a 5-fold excess of newly synthesized DNA over the DNA present in the original complex. The fractions isolated from the nuclear matrix of control Ehrlich ascite carcinoma cells and the cells incubated with novobiocin revealed quantitative and qualitative differences in the electrophoretic patterns of the proteins. Treatment of cells with novobiocin resulted in inhibition of DNA replicative synthesis and an increase in the protein/DNA ratio in the nuclear matrix.

The DNA polymerase activity of vaccinia virus 'virosomes': solubilization and properties.

Intracellular DNA-protein complexes ('virosomes') of vaccinia virus have been isolated. The solubilization of the 'virosome'-bound DNA polymerase activity was attempted by a variety of high-salt extraction procedures. The most efficient of these used 0.3 M-ammonium sulphate followed by brief sonication. The solubilized DNA polymerase activity from the 'virosomes', together with the DNA polymerases from 100000 g supernatant fluids from the cytoplasm of infected and uninfected cells were chromatographed on DEAE-cellulose and their properties compared. The 'virosome' DNA polymerase activity differed from the soluble vaccinia virus-induced DNA polymerase activity in its requirements for divalent cations and in respect of pH optimum, Km for the deoxyribonucleoside triphosphates and the effect of N-ethylmaleimide.