Differences of supranucleosomal organization in different kinds of chromatin: cell type-specific globular subunits containing different numbers of nucleosomes.

Fractions of homogeneously-sized supranucleosomal particles can be obtained in high yield and purity from various types of cells by brief micrococcal nuclease digestion (10 or 20 s) of condensed chromatin in 100 mM NaCl followed by sucrose gradient centrifugation and agarose gel electrophoresis. These chromatin particles, which contain only DNA and histones, differed according to cell type. Sea urchin spermatozoa (Paracentrotus lividus) gave rise to heavy particles (ca. 260 S) with a mean diameter (48 nm). These resembled the unit chromatin fibrils fixed in situ, which contain an average of 48 nucleosomes, as determined both by electron microscopy after unraveling in low salt buffer and gel electrophoresis. In contrast, higher order particles from chicken erythrocyte chromatin were smaller (105 S; 36-nm diam) and contained approximately 20 nucleosomes. The smallest type of supranucleosomal particle was obtained from chicken and rat liver (39 S; 32-nm diam; eight nucleosomes). Oligomeric chains of such granular particles could be recognized in regions of higher sucrose density, indicating that distinct supranucleosomal particles of globular shape are not an artifact of exposure to low salt concentrations but can be obtained at near-physiological ionic strength. The demonstration of different particle sizes in chromatin from different types of nuclei is contrary to the view that such granular particles are produced by artificial breakdown into "detached turns" from a uniform and general solenoid structure of approximately six nucleosomes per turn. Our observations indicate that the higher order packing of the nucleosomal chain can differ greatly in different types of nuclei and the supranucleosomal organization of chromatin differs between cell types and is related to the specific state of cell differentiation.

Visualization of nucleosomes in thin sections by stereo electron microscopy.

Nucleosomes (approximately diameter) were clearly visualized in thin sections (approximately 0.1 micrometer thick) of isolated chicken erythrocytes. The cells were lysed and fixed in low ionic strength buffers that maintained the chromatin as dispersed filaments and prevented the reformation of supranucleosomal structures. Stereo electron micrographs at high magnification demonstrate the stability of nucleosome structure in the dispersed chromatin state during fixation, dehydration, and embedding.

Higher order structure of simian virus 40 chromatin.

Simian virus 40 nucleoprotein complexes undergo an ionic strength-dependent structural transition. At moderate ionic strength they contain histone H1 as well as the nucleosomal histones and have a compact conformation with globular subunits 190 angstroms in diameter. At high ionic strength histone H1 is released, and the structure unfolds into chains with an average of 24 nucleosomes. The extended viral chromatin converts to the compact form by the addition of histone H1. Transcriptionally active simian virus 40 chromatin undergoes the same structural transitions. The higher order structure of viral chromatin may be analogous to the compact state of cellular chromatin fibers observed at physiological ionic strength.

Granzyme B is expressed in mouse mast cells in vivo and in vitro and causes delayed cell death independent of perforin.

Mast cells respond to pathogens and allergens by secreting a vast array of preformed and newly synthesized mediators, including enzymes, vasoactive amines, lipid mediators, cytokines and chemokines, thereby affecting innate and adaptive immune responses and pathogenesis. Here, we present evidence that skin-, but not lung-associated primary mast cells as well as in vitro-differentiated bone marrow-derived mast cells (BMMC) express granzyme (gzm) B, but not gzmA or perforin (perf). GzmB is associated with cytoplasmic granules of BMMC and secreted after Fcepsilon-receptor-mediated activation. BMMC from wild type but not gzmB-deficient mice cause cell death in susceptible adherent target cells, indicating that the perf-independent cytotoxicity of BMMC is executed by gzmB. Furthermore, gzmB induces a disorganization of endothelial cell-cell contacts. The data suggest that activated mast cells contribute, via secreted gzmB, to cell death, increased vascular permeability, leukocyte extravasation and subsequent inflammatory processes in affected tissues.

A novel karyoskeletal protein: characterization of protein NO145, the major component of nucleolar cortical skeleton in Xenopus oocytes.

The nucleolus is a ubiquitous, mostly spheroidal nuclear structure of all protein-synthesizing cells, with a well-defined functional compartmentalization. Although a number of nonribosomal proteins involved in ribosome formation have been identified, the elements responsible for the shape and internal architecture of nucleoli are still largely unknown. Here, we report the molecular characterization of a novel protein, NO145, which is a major and specific component of a nucleolar cortical skeleton resistant to high salt buffers. The amino acid sequence of this polypeptide with a SDS-PAGE mobility corresponding to M(r) 145,000 has been deduced from a cDNA clone isolated from a Xenopus laevis ovary expression library and defines a polypeptide of 977 amino acids with a calculated mass of 111 kDa, with partial sequence homology to a synaptonemal complex protein, SCP2. Antibodies specific for this protein have allowed its recognition in immunoblots of karyoskeleton-containing fractions of oocytes from different Xenopus species and have revealed its presence in all stages of oogenesis, followed by a specific and rapid degradation during egg formation. Immunolocalization studies at the light and electron microscopic level have shown that protein NO145 is exclusively located in a cage-like cortical structure around the entire nucleolus, consisting of a meshwork of patches and filaments that dissociates upon reduction of divalent cations. We propose that protein NO145 contributes to the assembly of a karyoskeletal structure specific for the nucleolar cortex of the extrachromosomal nucleoli of Xenopus oocytes, and we discuss the possibility that a similar structure is present in other cells and species.

Biological activities of a putative truncated hepatitis B virus X gene product fused to a polylysin stretch.

Truncated transcripts terminating within the HBx frame have been recognized previously in tumor and liver tissue of HCC patients. In this study biological activities of a predicted truncated HBx fused to a polylysin stretch (HBtx-polylysin) and of full length HBx were compared in NIH3T3 cells transfected with respective cDNA plasmids. Transactivation of a co-transfecting reporter gene and influence on neor DNA mediated transformation to G418 resistance were determined. In comparison to full length HBx the data indicate for HBtx-polylysin a lower transactivating capacity and as judged by the yield of colonies on a solid surface, a lower capacity to stimulate neor DNA mediated transformation. In soft agar the outgrowth into G418 resistant colonies was dependent on co-transfecting HBx cDNA. In providing this condition HBtx-polylysin had a much higher relative activity than full length HBx. Large cointegrants consisting of the plasmids carrying truncated HBx cDNA and neor DNA respectively were identified by chromosomal in situ hybridization. Based on Southern blot analyses extended concatemeres of the HBx cDNA plasmid constituted a main part of the cointegrants. Expression of truncated HBx cDNA was followed on the RNA and the protein level. The presence of this cDNA could be correlated to a compact spindle like cell appearance, its loss after prolonged passaging in the absence of G418 to a concomitant reversion to the phenotype of the NIH3T3 cell. Interspersed selection for G418 resistance stabilized the morphologically transformed phenotype. These results provide a basis to manipulate expression of truncated HBx and to recognize thereby processes leading to transformation.

Characterization of sequence elements involved in p53 stability regulation reveals cell type dependence for p53 degradation.

The growth suppressive properties of the tumor suppressor protein p53 are activated upon DNA damage. The activation of p53 is reflected in increased p53 levels which are, at least in part, the result of an extended half-life of the protein. Although this suggests that stabilization of p53 is an intrinsic feature of p53 activation, the mechanisms involved in p53 degradation and stabilization are poorly understood. Here we report on the identification of an internal deletion mutant of wild-type p53, termed delta62-96, which can be stably expressed in various cell lines. This deletion mutant has a turnover rate similar to wild-type p53 and its stability is upregulated by treatment with UV light. In cell lines that express endogenous mutant or no p53, however, delta62-96 appears to be stable, strongly indicating that these cell lines have lost the ability to degrade p53. Further characterization of delta62-96 by mutational analyses defines sequence and structural requirements for p53 degradation and indicates that none of the known p53 phosphorylation sites is essential with respect to p53 stability regulation upon UV-irradiation.

Uncoupling of p21WAF1/CIP1/SDI1 mRNA and protein expression upon genotoxic stress.

The p21WAF1/CIP1/SDI1 gene is an important regulator of crucial cellular processes, including cell cycle control, cellular differentiation, and the response to genotoxic stress. Induction of p21 gene expression upon DNA damage is widely believed to be p53-dependent. In the present study we analysed the expression of p21 following genotoxic stress, using different DNA-damaging agents and cellular systems. We found that the p21 response markedly varied between different cell lines and also for different genotoxic agents within the same cell line. Genotoxic induction of p21 mRNA expression can occur in the presence of p53-antagonists, such as overexpressed mdm-2 or human papillomavirus (HPV) E6, and in cells harbouring mutated p53 genes. Moreover, upon genotoxic stress, p21 mRNA and protein expression were found to be uncoupled in several cell lines. Thus, transcriptional and postranscriptional changes in p21 expression following DNA damage are not necessarily linked to the intracellular p53 status but strongly depend on the individual cellular background and the type of DNA-damaging agent. Our findings indicate that p21 expression following genotoxic stress underlies a complex control and can be substantially modulated on the posttranscriptional level in a cell-specific manner.

p53 overexpression is frequent in European hepatocellular carcinoma and largely independent of the codon 249 hot spot mutation.

Mutations in the p53 tumour suppressor gene have been recently described in hepatocellular carcinomas (HCC) from high risk areas such as China and South Africa. Our study was designed to assess the importance of p53 aberrations in HCCs from Europe, where the major risk factors in hepatocarcinogenesis, aflatoxin exposure and chronic hepatitis B virus (HBV) infection, do not play a dominant role. We investigated 22 HCCs and, as controls, their corresponding tumour-free liver tissues, seven livers with primary biliary cirrhosis and four morphologically normal livers. p53 overexpression, which is usually associated with point mutations of the p53 gene, was detected in 10 of the 22 HCCs by immunoblotting and immunohistochemistry. p53 expression was restricted to the nucleus in the positive cells, while all cells in the control tissues were negative. There was no obvious etiological preference in the p53 positive tumours. Particularly, underlying chronic HBV infection did not appear to be associated with an increased rate of p53 overexpression in European HCCs. SSCP and sequence analysis of exons 5-8 of the p53 gene revealed point mutations in six out of eight tumours with increased steady state levels of p53. In conclusion, our study demonstrates increased p53 levels due to point mutations in a significant proportion of European HCCs. The codon 249 mutation, which was detected in one of the cases, is not predominant in these tumours.

Interaction of DNA with DNA binding proteins. II. Displacement of Escherichia coli DNA unwinding protein and the condensed structure of DNA complexed with protein HD.

Three DNA binding proteins from Escherichia coli cells have been complexed with single-stranded phage fd DNA. Electron microscopy reveals granular substructures in the complexes formed with protein HD. In complexes of DNA unwinding protein with fd DNA both protein HD and phage-coded gene 5 protein partially displace the unwinding protein which results in the formation of structures characteristic for the DNA complexes formed with either protein HD or gene 5 protein alone. Combination of protein HD with double-stranded phage T7 DNA leads to a progressive folding and condensing of the genome. The structures observed are discussed in relation to current concepts of the packing of DNA in protein complexes.

Isolation, differential splicing and protein expression of a DNase on the human X chromosome.

A systematic search for genes differentially expressed in human tissues resulted in the isolation of a gene encoding a protein with high homology to DNase I. In addition to the recently described cDNA sequence (Parrish et al., 1995) we have isolated a transcript, alternatively spliced in the 5' noncoding region. The gene is located between the QM and the XAP-2 gene in Xq28 and encodes a 302 amino acid protein with 39% identity to human DNase I. Besides a high homology at the nucleotide and amino acid level, most exon-intron boundaries of DNase I and DNase X are identical, indicating that both genes may have evolved from a common ancestor. The predicted function was verified by expression of a recombinant protein in an inducible bacterial system and detection of DNase activity. In contrast to DNase I a 18 kdal amino terminal fragment of the full length 35 kdal protein exhibited DNase activity.

Mechanistic insights into p53-promoted RNA-RNA annealing.

The tumour suppressor protein p53 promotes the annealing of complementary nucleic acids in vitro. We observed an up to 1600-fold increase of RNA-RNA annealing by recombinant p53 protein which was shown to bind to RNA in sequence-independent way. Nuclease mapping experiments suggest that p53 binds to intramolecular duplex portions and only marginally changes the overall secondary structure of RNA at conditions of increased annealing. Thus, the mechanism of p53-promoted RNA-RNA annealing does not seem to be dependent on an activity that melts or changes RNA structure. The activation enthalpy of RNA-RNA annealing is decreased in the presence of p53, i.e. the p53 protein could stabilize the transition state whereas the activation entropy is unfavourable. A comparison with thermodynamic data measured for other facilitators strongly suggests that the mechanism of p53-promoted RNA-RNA annealing is distinct from the mechanism by which other facilitators work. The annealing activity of p53 is almost abolished in the presence of magnesium indicating that it can be sharply regulated in vitro and, in principle, could also be regulated in vivo.

A transcription enhancer acts in vitro over distances of hundreds of base-pairs on both circular and linear templates but not on chromatin-reconstituted DNA.

We have analyzed the effect of nucleosome formation and of the simian virus (SV40) enhancer on the efficiency of in vitro transcription. In a whole cell extract made from HeLa cells, nucleosome assembly on DNA results in the formation of chromatin-like complexes. However, transcription was detectable only when the DNA templates were partially or totally depleted of nucleosomes. On nucleosome-free templates, when the SV40 enhancer was present upstream from the complete SV40 early or rabbit beta-globin promoters, there was a five- to tenfold stimulation of specific transcription. When present upstream from its homologous promoter, the SV40 enhancer activated SV40 early transcription independently of its orientation with respect to the coding sequence. Point mutations known to impair the SV40 enhancer function in vivo had a similar effect in vitro. The extent of the enhancing effect was the same with linear or circular templates. When the SV40 enhancer was inserted upstream from the rabbit beta-globin gene, the activation of transcription was reduced with increasing distance between the enhancer and beta-globin upstream promoter elements, but was still significant over a distance of more than 400 base-pairs.

Visualization of large DNA molecules by electron microscopy with polyamines: application to the analysis of yeast endogenous and artificial chromosomes.

Standard visualization of nucleic acids by electron microscopy requires the use of special spreading techniques. The most common method takes advantage of the formation of a complex between negatively charged nucleic acid molecules and a positively charged monolayer film of proteins or cationic agents. Here, we describe an alternative protocol for the rapid visualization of DNA by electron microscopy based on the complexes formed when nucleic acids are exposed to buffers containing polyamines in the presence of sodium chloride. This procedure has been devised for the detection and analysis of large DNA molecules, such as yeast artificial chromosomes, but can be applied to DNA molecules of small size as well. The formation of DNA-polyamine complexes stabilizes large DNA molecules in solution and prevents shearing. This property allows large DNA molecules to remain intact after passage through microcapillaries used in the generation of transgenic mice by microinjection of fertilized eggs.

Isolation, characterization and chromosomal mapping of the mouse tyrosine aminotransferase gene.

The tyrosine aminotransferase (TAT) gene is expressed in a tissue and developmental-specific manner. In addition, this gene is regulated by glucocorticoid and polypeptide hormones and its expression is affected when a regulatory region near the albino locus of the mouse is deleted. In order to allow studies of the molecular effects of these deletion mutations we have isolated and characterized the mouse TAT gene. The gene is 9.2 x 10(3) bases in length and consists of 12 exons which give rise to a 2.3 x 10(3) base long messenger RNA. The DNA sequence at the 5' end of the gene was determined and compared with the corresponding sequence of the rat tyrosine aminotransferase gene. The sequence comparison showed extensive homology over the entire region sequenced. In addition, DNA: DNA heteroduplex studies between the mouse and rat tyrosine aminotransferase genes revealed that this homology extends over the entire gene and its flanking sequences. The mouse tyrosine aminotransferase gene has been mapped distal to the serum esterase-1 locus on mouse chromosome 8, using a restriction fragment length polymorphism between two mouse species. Since the albino deletions are located on mouse chromosome 7, the assignment of the TAT gene to chromosome 8 suggests that a regulatory factor(s) affecting TAT gene expression acts in trans.

Structural organization of the hepatitis B virus minichromosome.

The replicative intermediate of hepatitis B virus (HBV), the covalently closed, circular DNA, is organized into minichromosomes in the nucleus of the infected cell by histone and non-histone proteins. In this study we investigated the architecture of the HBV minichromosome in more detail. In contrast to cellular chromatin the nucleosomal spacing of the HBV minichromosome has been shown to be unusually reduced by approximately 10 %. A potential candidate responsible for an alteration in the chromatin structure of the HBV minichromosome is the HBV core protein. The HBV core protein has been implicated in the nuclear targeting process of the viral genome. The association of the HBV core protein with nuclear HBV replicative intermediates could strengthen this role. Our findings, confirmed by in vivo and in vitro experiments indicate that HBV core protein is a component of the HBV minichromosome, binds preferentially to HBV double-stranded DNA, and its binding results in a reduction of the nucleosomal spacing of the HBV nucleoprotein complexes by 10 %. From this model of the HBV minichromosome we propose that the HBV core protein may have an impact on the nuclear targeting of the HBV genome and be involved in viral transcription by regulating the nucleosomal arrangement of the HBV regulatory elements, probably in a positive manner.

Supranucleosomal organization of sea urchin sperm chromatin in regularly arranged 40 to 50 nm large granular subunits.

The organization of the chromatin of sperm nuclei of sea urchin (Parcentrotus lividus and Sphaerechinus granularis) was studied by electron microscopy using thin sections of fixed cells and spread preparations of chromatin obtained after various procedures involving incubation in low salt buffers. In fixed cells and in moderately dispersed preparations chromatin appeared in the form of fibrillar chains formed by closely apposed, distinctly sized granules that were larger than chromatin subunit structures so far described in other kinds of chromatin (mean diameters of particle units determined were 36 nm, in thin sections, and 47 nm, in spread preparations). Upon prolonged incubation in low salt buffer an increasing number of these large granular units was transformed into extended nucleosomal chains (nucleofilaments). Estimations indicated that the large granules contained 20 to 26 nucleosomes, i.e. 4.8 to 6.2 kb DNA, resulting in a linear DNA contraction ratio of 33 to 55, which is in a similar range as values reported for the supranucleosomal chromatin particle of the SV40 minichromosome but greater than contraction ratios of "superbead" structures. The unravelling of the large supranucleosomal granule into the extended nucleofilament chain in low salt buffers seemed to be a gradual process since intermediate stages of variable particles sizes and shapes were observed. This novel type of chromatin particle is considered to represent an example of a naturally occurring globular supranucleosomal packing unit of chromatin which is predominating, if not the only forms of supranucleosomal levels of chromatin organization and in relation to the special DNA content and histone composition of sea urchin sperm nucleosomes.

Reversible in vitro packing of nucleosomal filaments into globular supranucleosomal units in chromatin of whole chick erythrocyte nuclei.

Structural transitions between extended and condensed forms of chromatin have been studied by electron microscopy of whole chicken erythrocyte nuclei incubated in low buffer concentrations (0.5 mM sodium borate; 1 to 5 mM Tris-HCl) with and without additions of monovalent or divalent cations. Both spread preparations and ultrathin sections have been examined. It is shown that the condensed chromatin present in chicken erythrocytes is organized, at the supranucleosomal level, in granular particles (mean diameter 29 +/- 3 nM0 tightly apposed against each other in beaded chains (higher order fibrils). These chains of higher order granules are gradually unravelled in low salt buffers lacking divalent cations into extended nucleosomal filaments. When nuclear chromatin swollen in low salt buffer and extended uniformly into nucleosomal filaments is exposed to buffers containing higher concentrations of monovalent ions (e.g., 100 mM NaCl) or low concentrations of divalent cations (e.g., 0.05-0.5 mM MgCl2) rapid and almost complete recondensation of the entire chromatin of a nucleus into one compact spheroidal aggregate of chains of higher order granules (diameters 25-30 nm) is observed. This transition is reversible and whole nuclear chromatin can be induced, in vitro, to undergo several cycles of such conformational changes. The results show that reconstitution of globular forms of higher order packing of nucleosomes is not restricted to oligonucleosomal units but also takes place, in a rather uniform and regular mode, in long nucleosomal filaments as they are present in whole nuclei. The data are discussed in relation to other studies of chromatin ultrastructure and are interpreted to indicate that the predominant, if not exclusive form of supranucleosomal packing existing in condensed inactive chromatin of living cells is a chromatin fibril made up of serially arranged, close-packed granular particles ("suprabeads" sensu in [64]). In addition, the results suggest that higher order packing of chromatin into such supranucleosomal granules is not simply a function of ionic strength but that the type of ions present strongly influences the state of chromatin packing.

Coexistence of nucleosomal and various non-nucleosomal chromatin configurations in cells infected with herpes simplex virus.

Coexistence of four different forms of chromatin was observed by electron microscopy in nuclear spread preparations of monkey kidney cells during late stages of infection with herpes simplex virus (HSV-1 AMG). Besides typical nucleosomal (i) chromatin, thin (3-5 nm) strands morphologically indistinguishable from protein-free DNA were frequent, without (ii) or with (iii) sparse 10-22 nm large granules different from nucleosomes. In addition, uniformly thick (mean 17 nm), heavily stained chromatin strands (iv) were seen. The non-nucleosomal character of types (iii) and (iv) chromatin was also demonstrated by their resistance to histone removal in Sarkosyl and heparin. All four forms were seen in capsid-associated HSV-DNA molecules, and various combinations of these forms occurred in adjacent regions of the same DNA molecule, including the vicinity of replication branch points. Especially frequent were regions of chromatin types (ii) or (iii) alternating with thickly coated intercepts of type (iv) chromatin, the latter often displaying "bubble"-like strand separations. The appearance of chromatin types (ii)-(iv) was dependent on viral replication. These chromatin arrays were compared with structures observed in purified HSV-DNA from these cells. Patterns of single-stranded regions were found in HSV-DNA that were similar to those observed in the thickly coated type (iv) chromatin. It is concluded that, in these nuclei, non-nucleosomal arrangements can be formed, at least on viral DNA, under conditions of continued DNA synthesis and inhibited protein synthesis, and that single-stranded DNA is packed into a characteristic thick strand of non-nucleosomal chromatin by association with a special, probably virus-coded protein.

Cloning, bacterial synthesis, and characterization of immunoglobulin variable regions of a monoclonal antibody specific for the hepatitis B virus X protein.

The nucleotide (nt) sequences encoding the variable regions of the heavy (H) and light (L) chains were determined for a murine monoclonal antibody, 12/231/93, which is specific for a linear epitope located between amino acids 90 and 102 of the hepatitis B virus (HBV) X protein (HBx). The variable (V) regions of the H and L chains were shown to belong to the mouse H chain subgroup II (C) and kappa L chain group III, respectively. The cloned variable region sequences were used for the production of a Fab fragment in Escherichia coli, which had binding activity for membrane immobilized recombinant HBx. These gene sequences may be useful for the study of HBx function in cells that will support HBV replication.