Model studies of chromatin structure based on X-ray diffraction data.
(17/12332)
Model calculations are presented in order to interpret the X-ray diffraction diagrams given by chromatin gels. It is shown that by taking into account the hydration of chromatin subunits, the problem of calculating the interference function in concentrated gels is greatly simplified. In this way it is spossible to fully interpret the influence of concentration on the position and intensity of the various rings present in the X-ray diffraction patterns. The possibilities and limitations of models based on spherical symmetry are also discussed. It is concluded that each chromatin subunit most likely contains three turns of DNA in each 200 base pairs segment surrounding a central protein core. With the method presented here it is possible to test if other models of chromatin based on different kinds of evidence are compatible with the X-ray diffraction data. (+info)
Chromatin nu bodies: isolation, subfractionation and physical characterization.
(18/12332)
Monomer chromatin subunit particles (nu1) have been isolated in gram quantities by large-scale zonal centrifugation of micrococcal nuclease digests of chicken erythrocyte nuclei. nu1 can be stored, apparently indefinitely, frozen in 0.2 mM EDTA (pH 7.0) at less than or equal to 25 degrees C. Aliquots of the stored monomers have been subfractionated by dialysis against 0.1 M KCl buffers into a soluble fraction containing equimolar amounts of H4, H3, H2A, H2B associated with a DNA fragment of approximately 130-140 nucleotide pairs, and a precipitated fraction containing all of the histones including H5 and H1 associated with DNA fragments. The total nu1 and the KCl-soluble fraction of nu1 have been examined by sedimentation, diffusion, sedimentation equilibrium ultracentrifugation, low-angle X-ray diffraction, and electron microscopy. Physical parameters from all of these techniques are presented and correlated in this study. (+info)
Chromatin structure: a property of the higher structures of chromatin and in the time course of its formation during chromatin replication.
(19/12332)
The action of a number of enzymes and metals on one nuclear preparation were interpreted in terms of the existence of a fragile but highly DNAase-I resistant feature of chromatin superstructure. The generation of this DNAase-I resistance feature of chromatin was then followed during normal DNA synthesis in the regenerating rat liver by following the disappearance of a transitory DNAase-I susceptible state. This transitory, DNAase-I susceptible state appears to be extremely similar to the post-synthetic, DNAase-I susceptible state that has been described in He La32. (+info)
Nuclear foci of mammalian recombination proteins are located at single-stranded DNA regions formed after DNA damage.
(20/12332)
A sensitive and rapid in situ method was developed to visualize sites of single-stranded (ss) DNA in cultured cells and in experimental test animals. Anti-bromodeoxyuridine antibody recognizes the halogenated base analog incorporated into chromosomal DNA only when substituted DNA is in the single strand form. After treatment of cells with DNA-damaging agents or gamma irradiation, ssDNA molecules form nuclear foci in a dose-dependent manner within 60 min. The mammalian recombination protein Rad51 and the replication protein A then accumulate at sites of ssDNA and form foci, suggesting that these are sites of recombinational DNA repair. (+info)
Ligand-dependent activation of transcription in vitro by retinoic acid receptor alpha/retinoid X receptor alpha heterodimers that mimics transactivation by retinoids in vivo.
(21/12332)
All-trans and 9-cis retinoic acids (RA) signals are transduced by retinoic acid receptor/retinoid X receptor (RAR/RXR) heterodimers that act as functional units controlling the transcription of RA-responsive genes. With the aim of elucidating the underlying molecular mechanisms, we have developed an in vitro transcription system using a chromatin template made up of a minimal promoter and a direct repeat with 5-spacing-based RA response element. RARalpha and RXRalpha were expressed in and purified from baculovirus-infected Sf9 cells, and transcription was carried out by using naked DNA or chromatin templates. Transcription from naked templates was not affected by the presence of RA and/or RAR/RXR heterodimers. In contrast, very little transcription occurred from chromatin templates in the absence of RA or RAR/RXR heterodimers whereas their addition resulted in a dosage-dependent stimulation of transcription that never exceeded that occurring on naked DNA templates. Most importantly, the addition of synthetic agonistic or antagonistic retinoids to the chromatin transcription system mimicked their stimulatory or inhibitory action in vivo, and activation by a RXR-specific retinoid was subordinated to the binding of an agonist ligand to the RAR partner. Moreover, the addition of the p300 coactivator generated a synergistic enhancement of transcription. Thus, the dissection of this transcription system ultimately should lead to the elucidation of the molecular mechanisms by which RAR/RXR heterodimers control transcription in a ligand-dependent manner. (+info)
Male gametic cell-specific gene expression in flowering plants.
(22/12332)
The role of the male gamete-the sperm cell-in the process of fertilization is to recognize, adhere to, and fuse with the female gamete. These highly specialized functions are expected to be controlled by activation of a unique set of genes. However, male gametic cells traditionally have been regarded as transcriptionally quiescent because of highly condensed chromatin and a very reduced amount of cytoplasm. Here, we provide evidence for male gamete-specific gene expression in flowering plants. We identified and characterized a gene, LGC1, which was shown to be expressed exclusively in the male gametic cells. The gene product of LGC1 was localized at the surface of male gametic cells, suggesting a possible role in sperm-egg interactions. These findings represent an important step toward defining the molecular mechanisms of male gamete development and the cellular processes involved in fertilization of flowering plants. (+info)
Replication-dependent marking of DNA by PCNA facilitates CAF-1-coupled inheritance of chromatin.
(23/12332)
Chromatin assembly factor 1 (CAF-1) is required for inheritance of epigenetically determined chromosomal states in vivo and promotes assembly of chromatin during DNA replication in vitro. Herein, we demonstrate that after DNA replication, replicated, but not unreplicated, DNA is also competent for CAF-1-dependent chromatin assembly. The proliferating cell nuclear antigen (PCNA), a DNA polymerase clamp, is a component of the replication-dependent marking of DNA for chromatin assembly. The clamp loader, replication factor C (RFC), can reverse this mark by unloading PCNA from the replicated DNA. PCNA binds directly to p150, the largest subunit of CAF-1, and the two proteins colocalize at sites of DNA replication in cells. We suggest that PCNA and CAF-1 connect DNA replication to chromatin assembly and the inheritance of epigenetic chromosome states. (+info)
Histone ubiquitination and chromatin remodeling in mouse spermatogenesis.
(24/12332)
Male infertility in HR6B knockout mice is associated with impairment of spermatogenesis. The HR6B gene is a mammalian, autosomal homolog of the Saccharomyces cerevisiae gene Rad6 encoding a ubiquitin-conjugating enzyme. In addition, X-chromosomal HR6A has been identified, in human and mouse. RAD6 in yeast is required for a variety of cellular functions, including sporulation, DNA repair, and mutagenesis. Since RAD6 and its mammalian homologs can ubiquitinate histones in vitro, we have investigated the pattern of histone ubiquitination in mouse testis. By immunoblot and immunohistochemical analysis of wild-type mouse testis, a high amount of ubiquitinated H2A (uH2A) was detected in pachytene spermatocytes. This signal became undetectable in round spermatids, but then increased again during a relatively short developmental period, in elongating spermatids. No other ubiquitinated histones were observed. In the HR6B knockout mice, we failed to detect an overt defect in the overall pattern of histone ubiquitination. For somatic cell types, it has been shown that histone ubiquitination is associated with destabilization of nucleosomes, in relation to active gene transcription. Unexpectedly, the most intense uH2A signal in pachytene spermatocytes was detected in the sex body, an inactive nuclear structure that contains the heterochromatic X and Y chromosomes. The postmeiotic uH2A immunoexpression in elongating spermatids indicates that nucleosome destabilization induced by histone ubiquitination may play a facilitating role during histone-to-protamine replacement. (+info)