Replication origins are attached to the nuclear skeleton (original) (raw)
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Sites of Replication of Chromosomal DNA in a Eukaryotic Cell
Proceedings of the National Academy of Sciences, 1972
In mouse cells (line P815), newly synthesized DNA labeled for 20-30 sec during exponential growth is found by electron microscope autoradiography at sites throughout the cell nucleus. These sites are relatively more concentrated in the peripheral region of the nucleus (averaged over a random population of S-phase cells), probably reflecting a higher local concentration of DNA in this region. Newly synthesized DNA is not preferentially associated with purified nuclear envelopes, but is found in a fraction of the chromosomal deoxynucleoprotein whose buoyant density in CsCl after formaldehyde treatment is about 1% lower than that of the deoxynucleoprotein peak. Kinetics experiments suggest that this material is a precursor of mature deoxynucleoprotein; it may represent regions of deoxynucleoprotein containing replicating DNA and the additional proteins involved in DNA replication. Other complexes of newly replicated DNA that are found in the interphase after phenol extraction of nuclei...
In common with other eukaryotes, plant DNA is organised for replication as multiple replicons. A key event is the recognition of origins by the origin-recognition complex (ORC). Despite earlier indications, it is now likely that as in mammals, plant replication origins are not defined strictly by sequence; it is thus important to ascertain what features of origins are recognised by the ORC. Activation of origins occurs by stepwise loading of the pre-replication complex, all components of which have been identified in plants. This step is important in restricting DNA replication to once per cell cycle, although in plants, this restriction is relatively easily overcome, thus permitting DNA endoreduplication. It is also important to note the flexibility of origin use in relation to aspects of plant development. Is this flexibility mediated by ORC binding or at the pre-replication step? Following origin activation, the origin is prepared for initiation, again by the stepwise loading of ...
Mapping replicational sites in the eucaryotic cell nucleus
The Journal of cell biology, 1989
We have used fluorescent microscopy to map DNA replication sites in the interphase cell nucleus after incorporation of biotinylated dUTP into permeabilized PtK-1 kangaroo kidney or 3T3 mouse fibroblast cells. Discrete replication granules were found distributed throughout the nuclear interior and along the periphery. Three distinct patterns of replication sites in relationship to chromatin domains in the cell nucleus and the period of S phase were detected and termed type I (early to mid S), type II (mid to late S) and type III (late S). Similar patterns were seen with in vivo replicated DNA using antibodies to 5-bromodeoxyuridine. Extraction of the permeabilized cells with DNase I and 0.2 M ammonium sulfate revealed a striking maintenance of these replication granules and their distinct intranuclear arrangements with the remaining nuclear matrix structures despite the removal of greater than 90% of the total nuclear DNA. The in situ prepared nuclear matrix structures also incorpora...
Human origins of DNA replication selected from a library of nascent DNA
Molecular cell, 2005
laboratories, has shown that the S. cerevisae 11 bp 1 Molecular Medicine Laboratory consensus sequence is the target of the origin recogni-International Centre for Genetic Engineering tion complex (ORC) of proteins. The ORC complex and Biotechnology marks the origin site by sequence-specific interaction Padriciano, 99 throughout the cell cycle and directs the recruitment 34012 Trieste of other factors in a stepwise fashion, leading to the Italy formation of a prereplication complex (pre-RC; reviewed 2 Department of Cell Biology in Bell and Dutta, 2002). Tokyo Metropolitan Institute of Medical Science Given the conservation of most of the initiation fac-3-18-22 Honkomagome tors among different organisms and their functional Bunkyo-ku, Tokyo 113-8613 requirement for the DNA replication process itself, all Japan models of metazoan initiation of DNA replication are 3 University of Trieste largely based on the events occurring in S. cerevisiae. Faculty of Medicine Origins of DNA replication, however, are much less de-Piazzale Europa 1 fined in all other eukaryotic organisms, and the modes 34127 Trieste by which the initiation factors recognize or are recruited Italy to the origin sites are still to be discovered (Bielinsky and Gerbi, 2001; Gilbert, 2001). In S. pombe, origins are large (from 500-1500 bp in size) and apparently devoid Summary of defined consensus sequences, apart from long stretches of A-Ts (Dubey et al., 1994). The D. melano-The identification of metazoan origins of DNA replicagaster chorion locus origin is also dispersed and chartion has so far been hampered by the lack of a suitacterized by the presence of two distinct sequences: able genetic screening and by the cumbersomeness ori-β, which overlaps the replication origin, and a 440 of the currently available mapping procedures. Here bp amplification control element (ACE3), both of which we describe the construction of a library of nascent contain several ORC binding sites (Austin et al., 1999). DNA, representative of all cellular origin sequences, In higher eukaryotes, replication origins appear to be and its utilization as a screening probe for origin idenorganized either as initiation sites localized within a few tification in large genomic regions. The procedure dekb, as in the human Lamin B2 origin (Giacca et al., veloped was successfully applied to the human 1994), or even as multiple dispersed "initiation zones," 5q31.1 locus, encoding for the IL-3 and GM-CSF which might be the case of the origin downstream of genes. Two novel origins were identified and subsethe hamster DHFR gene (Vaughn et al., 1990). These quently characterized by competitive PCR mapping, apparent discrepancies highlight the lack of a suitable located 3.5 kb downstream of the GM-CSF gene. The number of origins to be compared: of the w30.000 oritwo origins (GM-CSF Ori1 and Ori2) were shown to gins of the human genome (DePamphilis, 1999), so far interact with different members of the DNA prerepliless than 30 have been identified and less than five cation complex. This observation reinforces the unicharacterized in molecular detail. In addition, only in a versal paradigm that initiation of DNA replication few instances has the mapping of the sites of initiation takes place at, or in close proximity to, the binding of DNA replication been correlated with the analysis of sites of the trans-acting initiator proteins. the interaction of some of the potential trans-acting factors (Abdurashidova et al., 2003; Keller et al., 2002; Introduction Ladenburger et al., 2002). During the last several years, a number of methods In the eukaryotic chromosomes, DNA is synthesized by have been developed to map origin activity within the multiple, tandemly organized replicons spaced bechromosomes. These methods are essentially based tween 5 and 100 kb; within each replicon, DNA replicaon the identification of structural or functional propertion starts at specific origins, which are activated only ties peculiar to the origin itself, such as the formation once per cell cycle, at a precisely defined time of the of small replication bubbles, the inversion of polarity of S-phase. In S. cerevisiae, origins of DNA replication are leading or lagging strands, or the issuing of short (w500 nt) stretches of nascent DNA (reviewed in: Dewell-defined sequences that show an intrinsic autono-Pamphilis, 1999; Giacca and Falaschi, 2002). None of mous replication activity and consist of an essential 11 these mapping procedures, however, appear ideal for bp consensus sequence plus several additional elethe long-range positioning of origins along the chromosomes. Here we describe the development of a novel *Correspondence: giacca@icgeb.org method for long-range mapping of origin sequences. 4 Present address: Department of Cell Biology, New York University, Similar to competitive PCR, this method, called sub-
Nuclear skeleton, DNA domains and control of replication and transcription
EJB Reviews 1991, 1991
Chromosomal DNA is organized in loops or domains of about 100 kb. Their ends seem to be attached to special protein skeletal structures. The DNA-attachment sites can be subdivided into permanent and transient types. The permanent or constitutive attachment sites, which are retained in all types of cells (including those inactive in replication and transcription), either coincide with or are located close to replication origins. This observation provides a simple way for isolation of DNA fragments containing replication origins. Such fragments from the chicken a-globin gene domain and other regions of the chicken genome contain DNA sequences which interact with nuclear proteins present in dividing cells, but absent from non-dividing cells. Several new consensus sequences interacting with nuclear proteins were detected. The 5' end region of the a-globin gene domain containing a replication origin was found to possess enhancer activity lacking tissue specificity. Hence, the domain organization of DNA is related to the organization of replication process. Other sets of data indicate that the integrity of DNA domains is important for maintaining transcription within the domain. According to these data, even a single nick at an distance of about 100 kbp seems to be sufficient for blocking transcription within the whole domain at the stage of RNA elongation. Thus, topological integrity of DNA may be an important factor involved in formation of active chromatin.
Chromosoma, 1990
To study whether specific DNA sequences are associated with nuclear membranes, residual DNA was extracted from DNase-treated nuclear envelopes prepared from erythrocytes of adult chickens (Gallus domesticus). This DNA was then blunt-end ligated into a bacterial plasmid vector. DNA blot analysis and nucleotide sequence determination revealed that approximately 30% of the cloned fragments consisted of different multiples of a 41 42 bp tandemly repeated, partially symmetrical sequence. In situ hybridization to chicken chromosomes demonstrated that the sequence was located primarily on microchromosomes, although some hybridization was also observed to macrochromosomes 7 and 8. Digestion of chicken DNA with any of a number of restriction enzymes did not completely reduce the intensity of a high molecular weight band to which the repeated sequence hybridized. These results, along with those obtained from in situ hybridization, suggested that many copies of this sequence are organized into large tandem arrays, and are not dispersed in many shorter repetitive blocks throughout the chicken genome. Although the repetitive sequence constituted approximately 10% of the chicken genome, it did not hybridize to quail or turkey DNA.
Journal of Cellular Biochemistry, 2009
The spatial organization of a 250 Kb region of chicken chromosome 14, which includes the alpha globin gene cluster, was studied using in situ hybridization of a corresponding BAC probe with nuclear halos. It was found that in non-erythroid cells (DT40) and cultured erythroid cells of definite lineage (HD3) the genomic region under study was partially (DT40 cells) or fully (HD3 cells) associated with the nuclear matrix. In contrast, in embryonic red blood cells (10-day RBC) the same area was located in the crown of DNA loops surrounding the nuclear matrix, although both globin genes and surrounding house-keeping genes were actively transcribed in these cells. This spatial organization was associated with the virtual absence of RNA polymerase II in nuclear matrices prepared from 10-day RBC. In contrast, in HD3 cells a significant portion of RNA polymerase II was present in nuclear matrices. Taken together, these observations suggest that in embryonic erythroid cells transcription does not occur in association with the nuclear matrix.