The histones of the insect trypanosomatid, Crithidia fasciculata.

The histone-like proteins of the monogenetic parasite Crithidia fasciculata were extracted with 0.2 M sulfuric acid either from purified nuclei, or from purified chromatin, in both cases in the presence of 1 mM tosyl lysylchloromethylketone and 2 mM phenyl methyl sulfonyl fluoride as proteinase inhibitors. The presence of histones in the flagellate, nonidentical with those from calf thymus used as controls, was shown by their electrophoretic patterns in three different polyacrylamide gel systems; their staining with Alkaline fast green, specific for basic proteins; their global amino acid composition and absorption spectrum and their molecular weights. The protein showing the slower mobility in SDS gels and the fastest mobility in the urea-acetic acid-Triton gels, seems to be an H1 histone, because of its metachromatic staining with Coomassie brilliant blue, solubility characteristics, differential destaining properties and amino acid composition. Band 5 in Triton-urea-acetic acid gels is probably an HMG protein. We conclude that C. fasciculata has a complete set of histones and that the lack of chromosome condensation during mitosis is not due to lack of histone H1.

Interactions of nucleic acids with fluorescent dyes: spectral properties of condensed complexes.

Interaction of cations with nucleic acids (NA) often results in condensation of the product. The driving force of aromatic cation-induced condensation is the cooperative interaction between ligand and single-stranded (ss) NA. This type of reaction is highly specific with regard to the primary and secondary structure of NA, and results in destabilization of the latter. The spectral properties of fluorescent intercalating and non-intercalating ligands [acridine orange, pyronin Y(G), DAPI, Hoechst 33258, and Hoechst 33342]-NA complexes were studied in both the relaxed and condensed form. The changes in absorption, excitation, and fluorescence emission spectra and fluorescence yield that followed the condensation were examined. Although some of these effects can be explained by changes in solvation of the fluorophore and its interaction with NA bases and the solvent, the overall effect of condensation on spectral properties of the complex is unpredictable. In particular, no correlation was found between these effects and the ds DNA binding mode of these ligands. Nevertheless, the spectral data associated with polymer condensation can yield information about the composition and structure of NA and can explain some nonspecific interactions of these probes.

Location of nucleosomes in simian virus 40 chromatin.

Over the past decade, the results of numerous indirect mappings analyses have not clarified whether or not nucleosomes occupy preferred positions in simian virus 40 (SV40) chromatin. To address this question more directly, we followed a shotgun cloning approach and determined the nucleotide sequences of over 400 cloned nucleosomal DNA fragments obtained from digestion of SV40 chromatin with micrococcal nuclease. Our results demonstrate and establish that nucleosomes do not occupy unique positions in SV40 minichromosomes and thus indicate the existence of at least several types of chromatin molecules having different nucleosome organization patterns. We developed two types of statistical analysis in order to examine the cloning data in greater detail. One type, overlap analysis, revealed the distribution of the cloned fragments with respect to SV40 DNA. The distribution exhibits an oscillating pattern, dividing the genome into regions of weak or strong nucleosome density. The other analysis determined the distribution of the midpoints of the cloned fragments and revealed potential strong and weak nucleosome location sites, and an early versus late distinction in organization of nucleosomes in SV40 chromatin. The late region appears to contain more strong nucleosome location sites (8) than the early region (4). The strongest nucleosome abuts the late side of the nuclease-hypersensitive region and includes the major transcription initiation site of the late genes. Another strong site precedes this nucleosome and includes sequences implicated in controlling the expression of the SV40 early and late genes. A strong or weak nucleosome location site is not apparent near the early side of the nucleosome-hypersensitive region. Only weak and overlapping nucleosome location sites are found in the region where replication terminates in the SV40 minichromosomes.

Seminal fluid from men with agenesis of the Wolffian ducts: zinc-binding properties and effects on sperm chromatin stability.

Zinc-binding properties were studied in 'prostatic fluid', i.e. in seminal plasma from patients with agenesis of the Wolffian ducts, and in split-ejaculate fractions dominated by seminal vesicular fluid. The effect of seminal fluid, with different zinc-binding properties, on the stability of zinc-dependent sperm chromatin was assessed by exposing sperm to 1% sodium dodecyl sulphate (SDS) for 60 min. Citrate was the only zinc ligand in 'prostatic fluid', as revealed by gel chromatography. Zinc in this fluid enhanced the stability of sperm chromatin. In contrast, the stability of sperm chromatin was decreased in seminal plasma dominated by vesicular fluid. These results are in accordance with the concept that prostatic fluid ensures the appropriate zinc content and stability of sperm chromatin, whereas abundance of vesicular fluid may jeopardize chromatin stability by reducing chromatin zinc content.

ADP-ribosylation of nuclear proteins in rat ventral prostate during ageing.

Poly(ADPR)polymerase activity and poly(ADP-ribosyl)ation of nuclear proteins have been investigated in ventral prostate nuclei of different aged rats (14, 28, 60, 180, 360 day old animals), by reverse-phase HPLC and acetic acid-urea polyacrylamide gel electrophoresis. The major ADP-ribose acceptor proteins were identified as histone H1 and H2b. It is concluded that concomitant with major changes to chromatin organization, poly(ADP-ribosyl)ation reaction is progressively inhibited during aging of rat ventral prostate. These results support the hypothesis that prostatic dysfunction in senescent animals is related to a failure of DNA repair mechanisms and deregulated template activity.

Repeat peptide motifs which contain beta-turns and modulate DNA condensation in chromatin.

In order to understand better the roles of repeating basic peptide motifs in modifying DNA structure, we have synthesized typical repeats found in the C-terminal domain of histone H1 (KTPKKAKKP)2 and in the N-terminal domain of nucleolin (ATPAKKAA)2. By using circular dichroism in conjunction with Raman and Fourier-transform infrared spectroscopies, we demonstrate that the abilities of the two peptides to affect DNA conformation are dramatically different. Whilst the binding of the nucleolin repeat to DNA does not significantly alter its conformation, the binding of H1 repeat induces a very marked DNA condensation, giving rise to a psi(-)-type circular dichroic spectrum. The H1 repeat thus adopts a more rigid beta-turn-containing structure which probably binds to the DNA minor groove as assessed by competition with the drug Hoechst 33258. Unexpectedly, the DNA condensation induced by the H1 repeat is enhanced by the nucleolin repeat which by itself does not promote any alteration in DNA conformation.

The appearance of hepatoma-associated chromosomal non-histone proteins in rat liver after a single dose of 3'-MDAB followed by treatment of phenobarbital.

Previously we have described polyclonal antibodies that recognized a group of nuclear nonhistone proteins whose molecular weights ranged in size from 170 to 220 kDa. These antigenic nonhistone chromosomal proteins are abundant in rat hepatoma chromatin. In this report we discuss the synthesis and cellular localization of these particular proteins during the multistage process of hepatocarcinogenesis. The appearance of these antigenic proteins in rat liver nuclei approximately parallels the appearance of alpha-fetoprotein in the cytosol of hepatocytes. However, the immunoreactivity of antigenic proteins increased steadily even during the prominent dip in the AFP concentration between 50 and 100 days of carcinogenesis. The effect of the tumor promoting agent, phenobarbital, on the synthesis of antigenic nuclear proteins was also studied. The appearance of hepatoma-associated non-histone chromosomal proteins at early stages of tumor promotion during hepatocarcinogenesis was observed. The results of these studies demonstrate that the hepatoma-associated non-histone proteins are expressed not only in hepatoma cells, but also in hepatocyte cells committed to carcinogenesis.

Accessibility of histone H1(0) and its structural domains to antibody binding in extended and folded chromatin.

The aim of this work was to study the accessibility of histone H1(0) and its structural domains to antibody binding in high molecular mass chromatin fragments of different conformations. Three types of specific antibody populations were used: (1) anti-H1(0) which reacted with antigenic determinants situated along the whole polypeptide chain, (2) anti-GH5 or anti-GH1(0) which recognized epitopes located in the globular region of H1(0) and (3) anti-C-tail antibodies reacting specifically with fragment 99-193 of the protein molecule. The immunoreactivity of the chromatin-bound antigen was investigated by solid-phase ELISA performed on glutaraldehyde-cross-linked chromatin and by an inhibition assay carried out with native chromatin in solution. The results of both methods were unidirectional and showed that: (1) the accessibility of H1(0) did not change with the compaction of the fiber; (2) the G-domain was not accessible to antibodies either in the relaxed or in the condensed state of the fragments, (3) the binding of the C-terminus-specific antibodies was different for isolated monosomes and for the chromatin fiber and (4) the degree of exposure of the epitopes of H1(0) in chromatin was much less than that of histone H1.

Chromosome-specific identification and quantification of S1 nuclease-sensitive sites in yeast chromatin by pulsed-field gel electrophoresis.

Sites that are sensitive to the single-strand-specific endonuclease S1 ('S1-sensitive sites', SSS) occur in native chromatin and, like DNA double-stranded breaks (DSB), they are induced by DNA-damaging agents, such as ionizing radiation. We have developed a method to quantify SSS and DSB in yeast chromatin by using pulsed-field gel electrophoresis (PFGE) to separate the intact chromosomal-length DNA molecules from the lower molecular-weight broken ones. Direct evaluation of the photonegatives of the ethidium bromide-stained gels by laser densitometry enabled us to calculate the numbers of DSB and SSS per DNA molecule. These numbers were determined from the bulk of the non-separated genomic DNA of yeast, corresponding to a single band in the PFGE (pulse time 10 seconds), and in each of the eight largest yeast chromosomes, corresponding to distinct bands in the PFGE gels (pulse time 50 seconds), which were not superimposed by the smear of the broken, low molecular-weight DNA. Furthermore, the induction of DSB and SSS in a specific chromosome (circular chromosome III) was determined by Southern hybridization of the PFGE gels with a suitable centromere probe, followed by densitometry of the autoradiographs. Our method allows the chromosome-specific monitoring of DSB and all those DNA structures that are processed either in vivo or in vitro into DSB and which may not be distributed randomly within the genome.

Structure of nucleosomes and organization of internucleosomal DNA in chromatin.

We have compared the mononucleosomal pattern produced by micrococcal nuclease digestion of condensed and unfolded chromatin and chromatin in nuclei from various sources with the repeat length varying from 165 to 240 base-pairs (bp). Upon digestion of isolated H1-containing chromatin of every tested type in a low ionic strength solution (unfolded chromatin), a standard series of mononucleosomes (MN) was formed: the core particle, MN145, and H1-containing, MN165, MN175, MN185, MN195, MN205 and MN215 (the indexes give an approximate length of the nucleosomal DNA that differs in these particles by an integral number of 10 bp). In addition to the pattern of unfolded chromatin, digestion of whole nuclei or condensed chromatin (high ionic strength of Ca2+) gave rise to nuclei-specific, H1-lacking MN155. Digestion of H1-lacking chromatin produced only MN145, MN155 and MN165 particles, indicating that the histone octamer can organize up to 165 bp of nucleosomal DNA. Although digestion of isolated sea urchin sperm chromatin (repeat length of about 240 bp) at a low ionic strength gave a typical "unfolded chromatin pattern", digests of spermal nuclei contained primarily MN145, MN155, MN235 and MN245 particles. A linear arrangement of histones along DNA (primary organization) of the core particle was found to be preserved in the mononucleosomes, with the spacer DNA length from 10 to 90 bp on one (in MN155) or both sides of core DNA being a multiple of about 10 bp. In MN235, the core particle occupies preferentially a central position with the length of the spacer DNA on both sides of the core DNA being usually about 30 + 60 or 40 + 50 bp. Histone H1 is localized at the ends of these particles, i.e. close to the centre of the spacer DNA. The finding that globular part of histones H3 and sea urchin sperm H2B can covalently bind to spacer DNA suggests their involvement in the organization of chromatin superstructure. Our data indicate that decondensation of chromatin is accompanied by rearrangement of histone H1 on the spacer DNA sites adjacent to the core particle and thus support a solenoid model for the chromatin superstructure in nuclei in which the core DNA together with the spacer DNA form a continuous superhelix.

Use of an immobilized enzyme and specific antibodies to analyse the accessibility and role of histone tails in chromatin structure.

Using limited proteolysis with subtilisin bound to collagen membranes, the degradation of the histone proteins revealed by specific antibodies was correlated to changes in chromatin conformation and condensation monitored by circular dichroism and electric birefringence. This new approach allows us to detect for the first time a hierarchy of histone tails cleavages. The terminal domains of H1, the NH2-terminal tail of H3 and the carboxy-terminal ends of histones H2A and H2B were found to be cleaved already at the early stages of proteolysis and this led to a decondensation of polynucleosomal chains. Thereafter the C-terminal part of H3 and both NH2-terminal regions of H2A and H2B became rapidly cleaved, resulting in relative reorientation of swinging nucleosomes or partially unfolded segments. Unexpectedly, this removal of tails of H1, H2B, H2A and H3 is not accompanied by significant changes in DNA-protein interactions resulting in free-oriented DNA. This might suggest that histone-histone interactions play a central role in stabilizing the solenoid.

Chromatin structure and gene expression in Alzheimer's disease.

Light micrococcal nuclease digestion was used to examine DNA associated with nucleosome populations isolated from Alzheimer's disease (AD) affected superior temporal lobe neocortical nuclei. 46.1% of the immediate 5' upstream DNA sequence of the single copy neurofilament light chain (NF-L) gene was found to be associated with a mononucleosome fraction in control neocortices. This fraction was reduced to 7.4% in age-matched AD-affected neocortex. No differences in accessibility to the nuclease probe was found between AD-affected and control temporal grey matter nuclei for the human prion HuPrP gene or for the NF-L gene in nuclei isolated from the primary visual cortex or the cerebellum. An AvaI restriction endonuclease site, located 124 base pairs upstream from the TATAA box in the NF-L leader sequence, was also found to be occluded in AD-affected nuclei. From this and previous data we conclude that within the AD-affected nucleus, focused changes in neuronal chromatin conformation occur. Increases in the packing density of chromatin may reduce transcription and alter the ability of neurons to generate sufficient levels of gene products to maintain normal neocortical function.

Characterization of a tandemly repeated DNA from the fleshfly Sarcophaga bullata.

In studies on the highly repetitive DNA sequences of the flesh fly Sarcophaga bullata, a 279 bp tandem repeat was cloned and sequenced. A 17 bp stretch within the clone was identical to a motif repeated five times in the satellite DNA of the Bermuda land crab. Southern DNA blotting showed the tandem repeat had a high degree of conservation of MboI sites, but had divergence for EcoRI sites; thus, all repeat units were not identical. The cloned DNA localized to the quinacrine-bright centromeric heterochromatin of the C and E autosomes and to sites on the chromosomal arms. In cases of asynapsis of homologs, the probe localized to euchromatic sites on both homologs or sometimes only on one homolog. The probe also localized near, to, or at a major developmental puff (B9). We conclude that blocks of this short interspersed repetitive DNA occur throughout the Sarcophaga genome in both heterochromatin and euchromatin, and also that the variable position of these sequences suggests they possess a degree of instability.

Analysis of Chlamydomonas reinhardtii histones and chromatin.

Chromatin spreads made from isolated nuclei of the unicellular green alga Chlamydomonas reinhardtii show the beaded fibers typical of eukaryotic polynucleosomes. Micrococcal nuclease digestions confirmed the presence of nucleosomes with a repeat length of 189 base pairs, essentially the same as typical mammalian cells. Basic nuclear proteins extracted from isolated nuclei or chromatin with 1 M calcium chloride and 0.3 M hydrochloric acid are resolved into seven major components by electrophoresis in the presence of sodium dodecyl sulfate (SDS). These seven components were subjected to qualitative peptide mapping with V8 protease on SDS gels for comparison with the major histone components of calf thymus. Finally, the C. reinhardtii basic nuclear proteins were fractionated by reversed phase high performance liquid chromatography and their amino acid composition determined. From these studies, we conclude that C. reinhardtii has a full complement of the five histones with properties very similar to those of both higher animals and higher plants.

Distribution pattern of acridine orange chromatin interaction products in rat glioma C6 cells at different phases of the cell cycle.

The purpose of the present study was to examine the distribution pattern of electron-dense acridine orange (AO) chromatin interaction products in rat glioma C6 cells at different phases of the cell cycle. For synchronization in the early S-phase the cells in logarithmic growth were treated with 3 micrograms/ml aphidicolin, a specific inhibitor of DNA polymerase alpha and then cultured in normal medium. For synchronization in the M-phase the cells cultured with aphidicolin and then returned to normal medium were treated with 0.05 micrograms/ml colcemid. Histoautoradiographic analysis of the C6 cells using the pulse chase method demonstrated approximately 16 h of cell cycle time and about 6.5 h of S-phase. Ultracytochemically, AO chromatin interaction products were found in all phases of the cell cycle except for the mitotic phase, namely in G1, S, and G2. The highest percentage of AO chromatin interaction products was observed in the early S-phase and the lowest in the G2 phase. The mean number of AO chromatin interaction products per nuclear area increased in the course of S-phase parallel with an increase of 3H-uridine uptake during the S-phase. The results show a characteristic distribution pattern of AO label specific for each of the four stages of the cell cycle, however, the significance of the coincident RNA synthetic activity remains to be elucidated.

Electron microscopic localization of acridine orange chromatin interaction products in rat pheochromocytoma PC12 cells.

The purpose of the present study is to examine the distribution pattern of acridine orange (AO) binding to DNA in rat adrenal pheochromocytoma PC12 cells that respond to nerve growth factor (NGF). PC12 cells were incubated in a medium containing 100 ng/ml of NGF for 1-14 days for AO ultracytochemistry. Electron microscopic studies revealed that AO binds to DNA exclusively within the euchromatin portion of the cell nucleus. About 35% of the untreated PC12 cells showed characteristic electron-dense interaction products within the nuclei. The average numbers of AO chromatin interaction products per cell nucleus and per micron 2 nuclear area were 45 and 1.6, respectively. In the presence of NGF, cell proliferation was suppressed. The cells gradually extend neurite-like cell processes. Mean 3H-labeling index was 40.0 +/- 2.2% in untreated cells and 13.0 +/- 2.3% in PC12 cells with NGF for 6 days after incubation for 30 min. With increasing the cellular differentiation percentages of AO positive cells and average numbers of AO chromatin interaction products per cell nucleus and per micron 2 nuclear area showed a progressive decrease. The results of the present and previous studies suggest that AO chromatin interaction products may be indicative of cell proliferation and differentiation.

Heterogeneous subgroups among malignant diffuse small B cell lymphomas. A combined nucleometric and immunocytologic study.

The degree of heterogeneity among subtypes was evaluated in 39 of the most frequent malignant, diffusely growing small B cell lymphomas by a combination of morphometry, automated image analysis, and immunocytologic techniques. Cluster analysis of nuclear profile parameters, including nuclear area, circularity factor, and chromatin distribution pattern, distinguished 3 groups. Each group was characterized by the preponderance of certain nuclear profile types, i.e. (a) small, rather regular (roundish) and dark staining (typical small lymphocytic lymphomas and immunocytomas); (b) small to intermediate size, rather regular (roundish) and pale staining (small to intermediate size variant); or (c) small to intermediate size, irregular, rather pale staining (diffuse follicular small cleaved cell (centrocytic) lymphomas and, probably, polymorphous immunocytomas). Group 3 was clearly distinct from the rest, but groups 1 and 2 also differed significantly. Every case displayed a mixture of the 13 registered nuclear profile types. Lymphoid cells with similar nuclear profile features occurred in B cell zones of non-neoplastic lymph nodes. Frequency distribution of nuclear profile parameters significantly differed from one lymphoma group to another. However, there were considerable overlaps. No clear correlation was found between nuclear profile types and immunophenotypes. Cellular surface antigen patterns showed an extensive intra- and inter-case variability. The findings support the notion of a certain individuality of malignant B cell lymphomas and of a marked heterogeneity among their subtypes.

Radial density distribution of chromatin: evidence that chromatin fibers have solid centers.

Fiber diameter, radial distribution of density, and radius of gyration were determined from scanning transmission electron microscopy (STEM) of unstained, frozen-dried chromatin fibers. Chromatin fibers isolated under physiological conditions (ionic strength, 124 mM) from Thyone briareus sperm (DNA linker length, n = 87 bp) and Necturus maculosus erythrocytes (n = 48 bp) were analyzed by objective image-processing techniques. The mean outer diameters were determined to be 38.0 nm (SD = 3.7 nm; SEM = 0.36 nm) and 31.2 nm (SD = 3.6 nm; SEM = 0.32 nm) for Thyone and Necturus, respectively. These data are inconsistent with the twisted-ribbon and solenoid models, which predict constant diameters of approximately 30 nm, independent of DNA linker length. Calculated radial density distributions of chromatin exhibited relatively uniform density with no central hole, although the 4-nm hole in tobacco mosaic virus (TMV) from the same micrographs was visualized clearly. The existence of density at the center of chromatin fibers is in strong disagreement with the hollow-solenoid and hollow-twisted-ribbon models, which predict central holes of 16 and 9 nm for chromatin of 38 and 31 nm diameter, respectively. The cross-sectional radii of gyration were calculated from the radial density distributions and found to be 13.6 nm for Thyone and 11.1 nm for Necturus, in good agreement with x-ray and neutron scattering. The STEM data do not support the solenoid or twisted-ribbon models for chromatin fiber structure. They do, however, support the double-helical crossed-linker models, which exhibit a strong dependence of fiber diameter upon DNA linker length and have linker DNA at the center.

Sequence-specific chemical modification of chromatin DNA with reactive derivatives of oligonucleotides.

Chemical modification of the chromatin DNA with alkylating derivatives of oligothymidylate (pT)16 and oligoadenylate (pA)16 bearing 4-(N-2-chloroethyl-N-methylamino)benzylphosphamide group at the 5'-phosphate has been investigated. It was found that the derivatives do react with DNA in chromatin. The reactions occur presumably at the complementary sequences of the DNA since the reaction of the oligothymidylate derivative is inhibited by oligonucleotide (pT)16 taken in excess and is not influenced by hexadecanucleotide of a random structure. Isolated DNA does not react with the oligothymidylate derivative. It is concluded that in chromatin, DNA is partially unwound or possesses some sites which can be opened easily in the presence of complementary oligonucleotides.