Structural-functional organization of DNA in the interphase nucleus. Structural aspects

The role of residual nuclear structures (structures persisting upon the treatment of nuclei with a non-ionic detergent, nucleases and 2 M NaCl) in the spatial organization of DNA in the interphase nucleus has been considered. Experimental works that have engendered the concept of loop level of DNA organization in the nucleus are discussed. A comparison is made of the loop-domain and rosette-like patterns of DNA organization in the interphase nucleus.     

Trout sperm chromatin. II. Ultrastructural aspects after salt dissociation of proteins

The ultrastructural organization of the trout sperm nucleus was studied in ultrathin sections and spread preparations after partial decondensation of the nucleus with increasing NaCl concentrations. The obtained results suggest that the organization of the trout sperm chromatin is much more complex than a pure nucleoprotamine. Three types of complexes were observed. The first one results from the association of DNA with protamines. This complex appears as a fibrous network when partially decondensed nuclei are digested with DNase I indicating that at least a part of DNA remains protected by protamines and favours models accepting a colinear alignment of the latter on the DNA molecules. The second type of structures represent the DNA-protamine fibers compacted into dense clumps which appear as separate compaction units seen upon partial decondensation of the sperm nucleus. A third type are complexes of the ring-shaped granular bodies tightly associated with DNA and resisting high salt-urea and detergent treatment.     

Preexisting nuclear architecture defines the intranuclear location of herpesvirus DNA replication structures.

Herpes simplex virus DNA replication proteins localize in characteristic patterns corresponding to viral DNA replication structures in the infected cell nucleus. The intranuclear spatial organization of the HSV DNA replication structures and the factors regulating their nuclear location remain to be defined. We have used the HSV ICP8 DNA-binding protein and bromodeoxyuridine labeling as markers for sites of herpesviral DNA synthesis to examine the spatial organization of these structures within the cell nucleus. Confocal microscopy and three-dimensional computer graphics reconstruction of optical series through infected cells indicated that viral DNA replication structures extend through the interior of the cell nucleus and appear to be spatially separate from the nuclear lamina. Examination of viral DNA replication structures in infected, binucleate cells showed similar or virtually identical patterns of DNA replication structures oriented along a twofold axis of symmetry between many of the sister nuclei. These results demonstrate that HSV DNA replication structures are organized in the interior of the nucleus and that their location is defined by preexisting host cell nuclear architecture, probably the internal nuclear matrix.     

Fine structural organization of a non-reticulate plant cell nucleus

We studied the fine structural organization of the meristematic nucleus in roots of Lycopesicon esculentum (tomato) using ultracytochemical and immunocytochemical approaches. The nucleus has a non-reticulate (i.e. low DNA content) structure whose supramolecular organization differs in some respects from that in reticulate nuclei, principally in the organization of the chromocentres associated with the nuclear envelope, with which centromeric structures appear to be associated. The main difference at the nucleolar level is found in the fibrillar centres, which have a low amount of DNA labelling and in which inclusions of condensed chromatin are present only very rarely. The distribution of nucleolar DNA amongst the nucleolar compartments is similar to that in reticulate nucleoli as demonstrated using an anti-DNA monoclonal antibody. Tomato nuclei have nucleolus-associated bodies or karyosomes, like other plant species with a low DNA content and non-reticulate nuclear organization. The nuclear ribonucleoprotein structures in the inter- and perichromatin regions, namely inter- and perichromatin fibrils and granules, show similar ultrastructural and cytochemical characteristics in both types of nuclei.Abbreviations NAC nucleolus associated chromatin - CES centromeric structures - NOR nucleolar organizing region - NAB nucleolus associated body - IG interchromatin granules - RNP ribonucleoprotein - Mab monoclonal antibody by M.F. Trendelenburg     

The synthesis and distribution of DNA in the macronucleus of Euplotes eurystomus

In an attempt to recognize any ordering of DNA synthesis which might occur in ciliates, the distribution of labelled DNA in the G₁ and S periods of the first, second, and third generations of a synchronized population of Euplotes eurystomus was studied by means of autoradiography. The results presented here show that the replicating DNA which at the time of label incorporation is restricted to a morphologically identifiable narrow region of the nucleus (the replication band), becomes dispersed and is evenly distributed throughout the nucleus. This dispersal of labelled DNA occurs previous to division and is observable throughout the G₁ period of the following generation. During the S phase of this second generation, this previously labelled DNA once again becomes restricted to a small portion of the nucleus. Now, however, it is present at the tips of the macronucleus independently of the position of the replication band. Again the labelled DNA is found to be dispersed during G₁ of the third generation. In the S period of this third generation however the radioactive DNA again appears localized in the replication bands which are found at the same position in the nucleus where they were when the pulse was given two generations earlier. The observations derived from the autoradiographic analysis suggest a non-permanent organization for at least those DNA molecules which are replicated during the first third of the S period. This DNA can be associated in either of two specific locations, replication band or tip, and these two patterns of organization alternate from generation to generation.     

Nucleus size and its effect on nucleosome stability in living cells

DNA architectural proteins play a major role in organization of chromosomal DNA in living cells by packaging it into chromatin, whose spatial conformation is determined by an intricate interplay between the DNA-binding properties of architectural proteins and physical constraints applied to the DNA by a tight nuclear space. Yet, the exact effects of the nucleus size on DNA-protein interactions and chromatin structure currently remain obscure. Furthermore, there is even no clear understanding of molecular mechanisms responsible for the nucleus size regulation in living cells. To find answers to these questions, we developed a general theoretical framework based on a combination of polymer field theory and transfer-matrix calculations, which showed that the nucleus size is mainly determined by the difference between the surface tensions of the nuclear envelope and the endoplasmic reticulum membrane as well as the osmotic pressure exerted by cytosolic macromolecules on the nucleus. In addition, the model demonstrated that the cell nucleus functions as a piezoelectric element, changing its electrostatic potential in a size-dependent manner. This effect has been found to have a profound impact on stability of nucleosomes, revealing a previously unknown link between the nucleus size and chromatin structure. Overall, our study provides new insights into the molecular mechanisms responsible for regulation of the nucleus size, as well as the potential role of nuclear organization in shaping the cell response to environmental cues.     

Nucleotides of rat liver cells: electron microscope analysis

Nucleoids of rat hepatocytes have been studied with the aid of electron microscopy. Proceeding from a morphologic comparison of nucleoids obtained at different conditions, it has been inferred that rosette-like structures are the basic element of nuclear DNA loop organization detected by sedimentation and fluorescent methods. Divalent metal ions play a significant role in stabilizing rosette-like structures. A scheme for DNA organization in the somatic cell interphase nucleus is suggested.     

On tracks and locomotives: the long route of DNA to the nucleus

Molecular motors have prominent functions in organelle transport, cytoskeletal organization, division and motility. The dyneins are one of the three families of cytoskeleton-based molecular motors and they travel along the cytoplasmic microtubule network towards the minus end of the microtubule. This directed movement is used by DNA viruses to deliver their infectious genome and proteins to the host cell nucleus. In recent studies, it has been hypothesized that Agrobacterium species use a similar pathway to deliver their infectious unit--a large complex between single-stranded DNA and proteins--to the host cell nucleus and that a karyophilic protein carrier that can deliver synthetic DNA to the nucleus is also driven by a dynein motor. These studies shed light on the mechanism of Agrobacterium-mediated genetic transformation and could lead to new methods for the efficient transfection of synthetic DNA.     

Architectural organization in the interphase nucleus of the protozoan Trypanosoma brucei: location of telomeres and mini-chromosomes.

We studied the spatial organization of chromatin in the interphase G1, S and G2 nucleus of the protozoan Trypanosoma brucei, applying in situ hybridization with conventional fluorescence and confocal scanning optical microscopy. The majority of the trypanosome telomere GGGTTA repeats from different chromosomes were found clustered together, either extending in a network through the nuclear interior or localized at the nuclear periphery. The population of one hundred mini-chromosomes was often asymmetrically located: either clustered in a narrow band in close association with the nuclear envelope or distributed into several clusters that segregated into roughly one half of the nucleus. The nuclear organization may undergo modifications during the cell cycle and development. We conclude that non-random spatial positioning of DNA exists in the nucleus of this protozoan. Finding a high level of structural organization in the interphase nucleus of T.brucei is an important first step towards understanding chromosome structure and functioning and its role in the control of gene expression.     

Fluorescence in situ hybridization to interphase cell nuclei in suspension allows flow cytometric analysis of chromosome content and microscopic analysis of nuclear organization

Summary Fluorescence hybridization to interphase nuclei in liquid suspension allows quantification of chromosome-specific DNA sequences using flow cytometry and the analysis of the three-dimensional positions of these sequences in the nucleus using fluorescence microscopy. The three-dimensional structure of nuclei is substantially intact after fluorescence hybridization in suspension, permitting the study of nuclear organization by optical sectioning. Images of the distribution of probe and total DNA fluroescence within a nucleus are collected at several focal planes by quantitative fluorescence microscopy and image processing. These images can be used to reconstruct the three-dimensional organization of the target sequences in the nucleus. We demonstrate here the simultaneous localization of two human chromosomes in an interphase nucleus using two probe labeling schemes (AAF and biotin). Alternatively, dual-beam flow cytometry is used to quantify the amount of bound probe and total DNA content. We demonstrate that the intensity of probe-linked fluorescence following hybridization is proportional to the amount of target DNA over a 100-fold range in target content. This was shown using four human/hamster somatic cell hybrids carrying different numbers of human chromosomes and diploid and tetraploid human cell lines hybridized with human genomic DNA. We also show that populations of male, female, and XYY nuclei can be discriminated by measuring their fluores-cence intensity following hybridization with a Y-chromosome-specific repetitive probe. The delay in the increase in Y-specific fluorescence until the end of S-phase is consistent with the results recorded in previous studies indicating that these sequences are among the last to replicate in the genome. A chromosome-17-specific repetitive probe is used to demonstrate that target sequences as small as one megabase (Mb) can be detected using fluorescence hybridization and flow cytometry.     

DNA oxidative damage in mammalian spermatozoa: where and why is the male nucleus affected?

Gamete DNA integrity is one key parameter conditioning reproductive success as well as the quality of life for the offspring. In particular, damage to the male nucleus can have profound negative effects on the outcome of fertilization. Because of the absence of repair activity of the quiescent mature spermatozoa it is easily subjected to nuclear damage, of which oxidative damage is by far the most prominent. In relation to the organization of the mammalian sperm nucleus we show here that one can correlate the nuclear regions of lower compaction with areas preferentially showing oxidative damage. More precisely, we show that oxidative DNA damage targets primarily histone-rich and nuclear matrix-attached domains located in the peripheral and basal regions of the mouse sperm nucleus. These particular sperm DNA domains were recently shown to be enriched in genes of paramount importance in postfertilization DNA replication events and in the onset of the embryonic developmental program. We propose that monitoring of sperm DNA oxidation using the type of assay presented here should be considered in clinical practice when one wants to estimate the integrity of the paternal nucleus along with more classical assays that essentially analyze DNA fragmentation and nucleus compaction.     

Nuclear and genome organization of Trypanosoma brucei

In this article, Klaus Ersfeld, Sara Melville and Keith Gull review current understanding of the structural organization of the nucleus of Trypanosoma brucei, and summarize recent data pertinent to the organization of its genome. Until recently, the cell biology of the trypanosome nucleus and issues of DNA organization and gene expression have often been treated as separate themes. However, recent work emphasizes the need for a more holistic approach to understanding these aspects of the biology of this parasite.     

Formation of DNA replication structures in herpes virus-infected cells requires a viral DNA binding protein

Eukaryotic DNA synthesis is thought to occur in multienzyme complexes present at numerous discrete sites throughout the nucleus. We demonstrate here that cellular DNA replication sites identified by bromodeoxyuridine labeling are relocated in cells infected with herpes simplex virus such that they correspond to viral prereplicative structures containing the HSV DNA replication protein, ICP8. Thus components of the cellular DNA replication apparatus are present at viral prereplicative sites. Mutant virus strains expressing defective ICP8 do not alter the pattern of host cell DNA replication sites, indicating that functional ICP8 is required for the redistribution of cellular DNA replication complexes. This demonstrates that a specific protein molecule can play a role in the organization of DNA replication proteins at discrete sites within the cell nucleus.     

Species-specific reorganization of the interphase chromosome architecture in generative tissue as a special type of chromosomal mutations associated with speciation

Epigenetic mechanisms of speciation are considered, including heterochromatic modifications and changes in spatial chromosome organization in the generative cell systems. The value of lamina, topoisomerase II, and a DNA polypurine tract in the attachment of chromosomes to the nuclear envelope is discussed. It is postulated that the main event leading to species-specific fixation of gene mutations, chromosomal mutations, and heterochromatin modifications in speciation is the rearrangement of spatial chromosome organization in the nucleus. The change in interchromosomal relationships associated with the reorganization of the system of chromosomal contacts with the nuclear envelope and the rearrangement of the chromocenter apparatus of the interphase nucleus is estimated as a systemic mutation directly related to speciation.     

Regulatory mechanisms of mammalian imprinting

Epigenetic modifications, such as monoallelic DNA methylation, covalent histone modifications, nonhistone proteins, chromatin folding, heterochromatinization, spatial nucleus organization are reviewed with regard to establishment and maintenance of imprinting in mammals. Special attention is paid to repeated DNA sequences as intermediates of the above epigenetic modifications. A suggestion is put forward relative to importance of preimplantation development, in particular, to chromosome organization and segregation in the establishment of imprinting. Some futher directions of imprinting mechanisms are also discussed.     

Compartmentalization of regulatory proteins in the cell nucleus

The cell nucleus is increasingly recognized as a spatially organized structure. In this review, the nature and controversies associated with nuclear compartmentalization are discussed. The relationship between nuclear structure and organization of proteins involved in the regulation of RNA polymerase II-transcribed genes is then discussed. Finally, very recent data on the mobility of these proteins within the cell nucleus is considered and their implications for regulation through compartmentalization of proteins and genomic DNA are discussed.